Non-pyrogenic preparation comprising nanoparticles synthesized by magnetotactic bacteria for medical or cosmetic applications

ABSTRACT

A non-pyrogenic preparation containing nanoparticles synthesized by magnetotactic bacteria for medical or cosmetic applications. The nanoparticles are constituted by a crystallized mineral central part including predominantly an iron oxide, as well as a surrounding coating without material from the magnetotactic bacteria.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/510,048, filed Mar. 9, 2017, which is a national phase application ofInternational Patent Application No. PCT/FR2016/000095, filed Jun. 15,2016, which claims priority to French Patent Application No. 1501267,filed Jun. 17, 2015, the entire contents of which are incorporatedherein by reference.

FIELD

The present invention relates to synthetic nanoparticles of bacterialorigin, modified, useful in medicine or in cosmetics.

BACKGROUND

Magnetotactic bacteria synthesize iron oxide nanoparticles, calledmagnetosomes, which possess outstanding properties due on the one handto the presence of a crystallized mineral central part of large size,typically of the order of 20 to 120 nm in diameter leading toadvantageous ferrimagnetic properties, and on the other hand to theirarrangement in chains that prevents magnetosome aggregation. Thesenanoparticles of bacterial origin display advantageous magneticproperties compared to nanoparticles resulting from chemical synthesis.For example, for an equivalent concentration in iron and when they areexposed to the application of an alternating magnetic field, suspensionsof magnetosome chains extracted from magnetotactic bacteria produce moreheat than chemical nanoparticles commonly used for magnetic hyperthermiasuch as chemical superparamagnetic iron oxide nanoparticles (SPION) andchemical ferrimagnetic iron oxide nanoparticles (FION). When suchsuspensions are administered to breast cancer tumors xeno-grafted underthe skin of mice and exposed to several applications of an alternatingmagnetic field, this induces anti-tumor activity (Alphandéry et al.,AcsNano, V. 5, P. 6279 (2011)). In addition, for an equivalentconcentration in iron, this anti-tumor activity is larger than thatobserved with SPION or HON.

However, these chains of magnetosomes come from gram negative bacteriaand can therefore possess in some cases a high endotoxin concentrationas well as biological material, which is difficult to preciselycharacterize. The present invention therefore aims to provide a methodof magnetosome production that enables to overcome these problems.

SUMMARY

The present invention results from the unexpected demonstration, by theinventors, that it was possible to replace the natural surroundingcoating of the magnetosomes with another coating to yield non-pyrogenicsynthetic nanoparticles, which can be used in health, diagnosis and/orcosmetics.

Thus, the present invention relates to a preparation comprising at leastone synthetic nanoparticle, wherein the nanoparticle comprises:

-   -   A crystallized mineral central part comprising predominantly an        iron oxide, synthesized by a living organism, and    -   A surrounding coating comprising no material originating from        the living organism.

The preparation according to the invention can comprise at least 1, 2,3, 5, 10, 100, 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ synthetic nanoparticlescomprised in a volume of 1 μm³, 1 mm³, or 1 dm³. It may be a suspensionof synthetic nanoparticles, preferentially comprised in a liquid medium,or it may be an assembly of synthetic nanoparticles comprised in aliquid, solid or gaseous medium.

The present invention also relates to a crystallized central partpredominantly comprising an iron oxide synthesized by a living organism,and not comprising any coating.

A synthetic nanoparticle is a particle whose size is in at least onedimension lower than 10, 5, 2, 5 or 1 μm, preferentially lower than 750,500, 400 or 300 nm, most preferentially lower than 200 or 100 nm, whichis preferentially in a solid state and whose size is notably measurableby transmission electron microscopy (TEM). The word “synthetic”indicates that the nanoparticle has been manufactured using at least onestep involving man, in particular using biological or chemicalprocesses.

The synthetic nanoparticle according to the invention can possess atleast one magnetic property, such as:

-   -   be diamagnetic, superparamagnetic, preferentially ferrimagnetic        or ferromagnetic,    -   possess at least one magnetic domain,    -   possess at least one non-zero magnetic moment,    -   possess a coercivity larger than 0.5, 1, 10, 100, or 1000 Oe at        a temperature above 0, 10, 100, 273, 310, 373, or 1000 K, or    -   possess a ratio between remanant and saturating magnetization,        which is larger than 0.01, 0.1, 0.2, 0.5, 0.7, 0.9, 0.95, or        0.99 at temperature larger than 0, 10, 100, 273, 310, 373, or        1000 K, these properties being preferentially due to the        properties of its central part.

The synthetic nanoparticle according to the invention is preferentiallya nanoparticle possessing a single magnetic domain, which can lead tobetter magnetic properties compared with nanoparticles having severalmagnetic domains.

The synthetic nanoparticle according to the invention is preferentiallya ferrimagnetic or ferromagnetic nanoparticle, in particular ananoparticle whose magnetic moment is thermally stable at physiologicaltemperature and/or whose size is larger than 1, 2, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200, 500 or 1000 nm.

The synthetic nanoparticle according to the invention may be asuperparamagnetic nanoparticle, in particular a nanoparticle whosemagnetic moment is thermally unstable at physiological temperatureand/or whose size is lower than 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 70, 80, 90, 100, 150, 200, 500 or 1000 nm.

The average diameter of the synthetic nanoparticle according to theinvention can be measured using transmission electron microscopy (TEM)or with the help of a dynamic or non-dynamic light scattering method.

The central part of the synthetic nanoparticle predominantly comprisesan iron oxide, i.e. at least 10 or 25%, preferentially at least 50, 75,95, 97, 99, 99.5 or 99.9% of oxide of iron. This percentage of ironoxide corresponds in particular to the number of atoms comprised in theiron oxide of the central part divided by the number of atoms comprisedin the iron oxide of the synthetic nanoparticle or to the mass of ironoxide comprised in the central part divided by the mass of iron oxidecomprised in the synthetic nanoparticle.

Preferentially, the iron oxide is maghemite, magnetite or anintermediate composition between maghemite and magnetite.

Preferentially, the living organism synthesizing the central part ofsynthetic nanoparticles according to the invention is a eukaryotic cellor a bacterium, more preferentially a bacterium synthesizing magneticnanoparticles, and even more preferentially a magnetotactic bacterium.Most preferentially, the bacteria according to the invention belong tothe magnetotactic strains, in particular Magnetospirillum magneticumAMB-1, Magnetococcus marinus sp. MC-1, Magnetovirrium blakemorei MV-1,MV-2 and MV-4, Magnetospirillum magnetotacticum MS-1, Magnetospirillumgryphiswaldense MSR-1, Magnetospirillum magneticum MGT-1, andDesulfovibrio magneticus RS-1.

One will also notice that the synthetic nanoparticle according to theinvention differs from the magnetosomes extracted from the magnetotacticbacteria, usually arranged in chains, by the absence of organic materialoriginating from magnetotactic bacteria in their coating. The coatingpreferentially does not comprise non-denatured organic material.Denaturation is defined here as the loss, by a biological macromolecule(e.g. nucleic acid or protein), of its usual three-dimensionalconformation. The synthetic nanoparticle may indeed comprise, in itscoating, denatured organic matter originating from magnetotacticbacteria. This denatured organic material may appear after a treatmentintended to purify nanoparticles, such as a chemical, thermal ormagnetic treatment or a combination of these treatments.

In one embodiment, the coating comprises less than 50%, 25%, 10%, or 1%of non-denatured organic material coming from magnetotactic bacteria,e.g. lipids, endotoxins and/or non-denatured proteins coming frommagnetotactic bacteria. This percentage of non-denatured organicmaterial corresponds in particular to the mass of this materialcomprised in the coating divided by the mass of the syntheticnanoparticle. This small quantity of organic material coming frombacteria can be characterized by a mass of chemical functional groups atthe surface of or inside the purified central part, such as phosphatesor amines, which is less than 1 mg, 0.1 mg, 0.01 mg, 0.001 mg, 0.0001 mgor 40 ng per 1 mg in total iron of the central part. This low quantityof non-denatured organic material can enable to produce a suspension ofsynthetic nanoparticles with a sufficiently low level of pyrogenicityaccording to the standards in force, in particular for allowingadministration to a human or an animal.

In one embodiment, the preparation according to the invention comprisesat least 2, 5, 10, 20, 30, 50, 100, 150, 200, 500, 10000, 50000 or100000 synthetic nanoparticles which are bond to each other byconnecting material. The connecting material may have at least one ofthe following properties: i), be made, at least in part, of the samematerial(s) as that(those) of the coating of the syntheticnanoparticles, ii), link at least two synthetic nanoparticles together,over a distance lower or larger than 1 mm, 100 μm, 10 μm, 1 μm, 100 nm,50 nm, 40 nm, 30 nm, 20 nm, 10 nm, 5 nm, 2 nm, or 1 nm, iii), promotecellular internalization, iv), be positively or negatively charged or beneutral, v), have or not have a therapeutic or diagnostic effect, vi),be sufficiently resistant to ensure the link between at least twosynthetic nanoparticles either when the preparation is in a liquid,physical, gaseous, or biological medium, or after sterilization or aftera physical, biological, or chemical treatment, for example with the helpof the solution or medium used for the formulation of the preparationnotably for the administration to an organism, vii), comprise less than20%, 15%, 10%, 5%, 2%, 1%, 0.1%, 0.01%, or 0.001% of carbonaceousmaterial coming from the living organism, viii), not come from theliving organism.

The synthetic nanoparticles may be arranged in a geometric figure whichcan be: a straight chain, a circle, a square, a rectangle, a triangle, apentagon, a hexagon, a heptagon, an octagon, a polygon or a polyhedron.These figures can be observed by Transmission Electron Microscopy.

An arrangement of at least two synthetic nanoparticles according to ageometric figure can correspond to an arrangement of these nanoparticleshaving: i), crystallographic axes orientated in a preferentialdirection, ii), at least two sides or faces belonging to two differentsynthetic nanoparticles, which are parallel or perpendicular, or whichform an angle larger or lower than 1, 2, 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 60, 70, 80, 90, 100, 150, 180, 225, 250, 300, 325, 350, or 379degrees, iii), an alignment parallel to a magnetic field when thesenanoparticles are exposed to the application of a magnetic field, inparticular a field of larger intensity than 10⁻¹⁰ T, 10⁻⁹ T, 10⁻⁸ T,10⁻⁷ T, 10⁻⁶ T, 10⁻⁵ T, 10⁻⁴ T, 10⁻³ T, 10⁻² T, 10⁻¹ T, 1 T or, iv),linking materials linking at least two synthetic nanoparticles together.

An arrangement of at least two synthetic nanoparticles within ageometric figure can be characterized by a variation of the zetapotential as a function of pH, which is a continuous variation such as acontinuous decrease or increase, preferentially a continuous decrease,when the pH increases from 1 to 14, 2 to 12, 3 to 11, 4 to 10, 5 to 9,or 6 to 8, wherein a continuous decrease or increase may correspond to avariation measured between more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 different pH units which is either a decrease or an increase.

In contrast, an arrangement of at least two synthetic nanoparticles,which is not in a geometric figure, such as an aggregate, can becharacterized by a variation of zeta potential as a function of pH,which is a succession of at least 1, 2, 3, 4, 5, 7, 9, 10, 12, 15, 20decrease(s) and increase(s) when the pH increases from 1 to 14, 2 to 12,3 to 11, 4 to 10, 5 to 9, or 6 to 8.

An arrangement within a geometrical figure may take place in a solid,liquid or gaseous medium, i.e. in particular in a medium surrounding thesynthetic nanoparticles, such as the medium used for the formulation ofthe synthetic nanoparticles or that surrounding these nanoparticlesduring their studies or characterizations.

According to the invention, it is preferred that when the livingorganism synthesizing the central part of the synthetic nanoparticles isa magnetotactic bacterium, the synthetic nanoparticles according to theinvention comprise the central parts of the magnetosomes coated with acompound.

Preferentially, the central part of the synthetic nanoparticlesaccording to the invention has at least one of the following properties:

-   (α), a crystalline part presenting at least 1, 2, 5, 10, 50, 100,    150 or 200 crystalline planes, being preferentially within a mineral    form, comprising predominantly iron oxide, preferentially maghemite,    notably when the central part is brought into contact with oxygen,    magnetite, notably when the central part is not brought into contact    with oxygen, or a mixture of maghemite and magnetite,-   (β), a ferrimagnetic or ferromagnetic behavior, notably at    physiological temperatures,-   (χ), a single magnetic domain, or magnetic monodomain,-   (δ), a magnetic microstructure, which can be characterized by the    presence of magnetic field lines that can be orientated in a    preferential direction such as an easy magnetization axis or a    crystallographic direction of the central part of the synthetic    nanoparticles such as [111], where such magnetic microstructure may    under certain conditions be observable, notably by electronic    holography,-   (ε), a size between 1 nm and 2 μm, 5 nm and 1 μm, 5 and 500 nm, 5    and 250 nm, 5 and 100 nm, 5 and 80 nm, 5 and 60 nm, 10 nm and 1 μm,    10 and 500 nm, 10 and 250 nm, 10 and 100 nm, 10 and 80 nm, 10 and 60    nm, 15 nm and 1 μm, 15 and 500 nm, 15 and 250 nm, 15 and 100 nm, 15    and 80 nm, 15 and 60 nm, 20 nm and 1 μm, 20 and 500 nm, 20 and 250    nm, 20 and 100 nm, 20 and 80 nm or 20 and 60 nm,-   (ϕ), a size larger than 1, 2, 5, 10, 15, 20, 25, 30, 35 or 40 nm,-   (γ), a size lower than 2000, 1000, 500, 400, 300, 200, 150, 120,    100, 95, 90, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15,    10 or 5 nm,-   (η), the possible presence of a zeta potential, a charge, a surface    charge,-   (τ), an isoelectric point, preferentially acidic of pH lower than or    equal to 7, 6, 5, 4, 3, 2 or 1,-   (u), an isoelectric point, which is the closest to 7 when there    remains the lowest quantity of organic material at the surface of    the central part; when there remains a low quantity of organic    material, the isoelectric point can be between pH 6.5 and pH 7.5,    between pH 6.2 and pH 7.1, or between pH 6.1 and pH 7.1.

As will be understood by the person skilled in the art, the zetapotential, the charge, the surface charge usually depend on the pH, thesalinity, the bacterial species synthesizing the central part of thesynthetic nanoparticles, the aggregation state, the percentage oforganic material at the surface of these central parts. They may be:(1), positive at acid pH, at pH equal to or lower than 7, 6, 5, 4, 3, 2,or 1, (2), negative at basic pH, at pH larger than or equal to 13, 12,11, 10, 9, 8, 7, (3), vary as a function of pH within a range comprisedbetween −10 and 10 mV, −20 and 20 mV, −30 and 30 mV, −40 and 40 mV, −50and 50 mV, −60 and 60 mV, −70 and 70 mV, −80 and 80 mV, −90 and 90 mV,−100 and 100 mV, −150 and 150 mV, or −200 and 200 mV, (4), vary the mostsignificantly as a function of pH values when the quantity of organicmaterial, preferentially of carbonaceous material, remaining at thesurface of the central parts of these synthetic nanoparticles, is thelowest.

Preferentially, the central part of the synthetic nanoparticlescomprises less than 50, 25, 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.01 or0.001% organic material. As used herein, this percentage of organicmaterial represents the percentage in mass or in number of atoms or involume of any chemical element or combination of chemical elementscomprised in the organic material such as carbon, nitrogen, phosphorus.This percentage can be measured using instruments of elementary analysissuch as total organic carbon (TOC) or carbon, hydrogen, nitrogen,sulfur, oxygen (CHNS/O) analyzers.

In one embodiment, the coating according to the invention, alsodesignated as surrounding coating, is a solid, liquid or gaseousmaterial, preferentially in the solid state, which surrounds the centralpart, notably in the form of a layer, crystallized or not, notably overa distance measured from the center of the central part which is lowerthan 100 μm, 10 μm, 5 μm, 1 μm, 500 nm, 250 nm, 100 nm, 10 nm, or 1 nm.This coating can in particular serve to stabilize the central part or tolink the synthetic nanoparticles together.

The coating may have at least one property in common with the centralpart or at least one property different from the central part.

According to the invention, the surrounding coating preferentially doesnot comprise non-denatured material originating from the livingorganism. It may comprise less than 50, 40, 30, 20, 10, 5, 4, 3, 2, 1,0.1, 0.05, 0.01, 0.001 or 0.0001% of this material. This percentage canbe defined as the ratio between the number of atoms coming from theliving organism comprised in the coating divided by the total number ofatoms comprised in the coating or the ratio between the mass of atomscoming from the living organism comprised in the coating divided by thetotal mass of atoms comprised in the coating. This percentage cancorrespond to a percentage of carbonaceous or organic material.

The synthetic nanoparticle, according to the invention, isnon-pyrogenic, wherein non-pyrogenicity can be defined by measuring thequantity of endotoxins per milligram of synthetic nanoparticle,preferentially comprised in 1 milliliter of the preparation, which islower than or equal to 10⁹, 10⁸, 10⁷ or 10⁶, preferentially lower thanor equal to 10⁴ or 10³, most preferentially lower than or equal to 100,50, 10, 5 or 1 endotoxin unit (EU). As the man skilled in the art knows,1 EU can correspond to a quantity of 100 μg per mL of endotoxinsoriginating from E-Coli.

Preferentially, the synthetic nanoparticle is non-pyrogenic when itscoating or its central part is non-pyrogenic, preferentially when itscoating and its central part are non-pyrogenic, where non-pyrogenicitycan in this case be defined by measuring the quantity of endotoxins permilligram of the central part and/or of the coating of the syntheticnanoparticle, preferentially comprised in 1 milliliter of thepreparation, which is lower than or equal to 10⁹, 10⁸, 10⁷ or 10⁶,preferentially lower than or equal to 10⁴ or 10³, most preferentiallylower than or equal to 100, 50, 10, 5 or 1 endotoxin unit (EU).

As used herein, a preparation is non-pyrogenic when substances otherthan the synthetic nanoparticles comprised in the preparation, such asthe substances comprised in the excipient and/or the solvent of thepreparation and/or the matrix surrounding the synthetic nanoparticlesare non-pyrogenic.

Preferentially, the non-pyrogenic preparation is characterized by apercentage of endotoxins lower than or equal to 50, 20, 1, 10⁻, 10⁻²,10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸ or 10⁻⁹%, where this percentage canbe: (i), the mass of endotoxin divided by the total mass of thepreparation, (ii), the volume occupied by the endotoxins divided by thetotal volume of the preparation, (iii), the number of atoms comprised inthe endotoxins of the preparation divided by the total number of atomscomprised in the preparation.

Preferentially, the non-pyrogenic preparation is characterized by aquantity of endotoxins lower than or equal to 10⁹, 10⁸, 10⁷ or 10⁶,preferentially lower than or equal to 10⁴ or 10³, most preferentiallylower than or equal to 100, 50, 10, 5 or 1 EU or EU per milligram ofiron oxide or EU per milliliter or EU per milligram of iron oxide permilliliter.

In one embodiment, the non-pyrogenic preparation is a drug administeredto an organism such as an individual or an animal. It preferentiallycomprises an endotoxin quantity lower than: (i), 5 EU per kilogram ofbody weight of this organism per hour of administration fornon-intrathecal administration and, (ii), 0.2 EU per kilogram of bodyweight of this organism per hour of administration for intrathecaladministration.

In one embodiment, the non-pyrogenic preparation is a medical device, inparticular an invasive device. It then preferentially comprises anendotoxin quantity, preferentially measured at the surface of themedical device, which is lower than: (i), 0.5 EU per milliliter of thepreparation when the latter is not in contact with the cerebrospinalliquid and, (ii), 0.02 EU per milliliter of the preparation when thepreparation is in contact with this liquid.

Preferentially, the non-pyrogenic preparation according to the inventioncomprises an endotoxin quantity, which abides by the applicableregulatory standards applicable to drug, medical device or cosmeticproduct, in particular abides by the pharmacopoeia, most preferentiallythe European and/or American pharmacopoeia(s).

The non-pyrogenic preparation can optionally be administered to anorganism within an administration time larger than or equal to thefollowing values: 1, 30 or 60 second(s), 1, 30 or 60 minute(s), 1, 15 or24 hour(s), 1 or 5 day(s), 1 or 4 week(s). In case where thenon-pyrogenic preparation is administered to an organism within anadministration time larger than or equal to these values, the quantityof endotoxins comprised in the preparation may be larger than when thesame preparation is administered within a time, which is lower thanthese values.

Preferentially, the quantity of endotoxins in the preparation ismeasured by a limulus amoebocyte lysate test (LAL). As it will beunderstood by the person skilled in the art, one will prefer to ensurethat the nanoparticles do not interfere with the test, in particular bymeasuring a recovery rate. This rate can be defined as being equal toC_(total)/C₁+C₂, where C_(total) is the endotoxin concentration ofsuspensions of synthetic nanoparticles mixed with a known quantity ofendotoxins of e.g. 0.5 EU/mL. In this example, C₁ is the concentrationof endotoxins in the different suspensions of synthetic nanoparticlesand C₂=0.5 EU/mL. It can be considered that there is no interferencewhen this recovery ratio is larger than or equal to 10 or 30,preferentially 50, 100 or 150%.

The pyrogenicity of the preparation according to the invention may beevaluated by a rabbit pyrogenicity test, in particular according to ISO10993-11, chapter 151 of the American Pharmacopoeia or the EuropeanPharmacopoeia. This test can be carried out by administering thepreparation intravenously to 1 or several rabbits, preferentially 3,preferentially using a quantity of synthetic nanoparticles, which istypically but not necessarily larger than or equal to 1 mg. Inparticular, in the case where the preparation is a medical device, sucha quantity appears to be suitable, since it is larger than 6 cm² per mlor mg of synthetic nanoparticles, in particular recommended by ISO10993-12 standard. Indeed, considering the surface of cubic syntheticnanoparticle with a side of 50 nm, which is 15.10⁻¹¹ cm², and that 1 mgof synthetic nanoparticle composed predominantly of maghemite comprises1.6 10⁻¹² synthetic nanoparticles, it can be estimated that 1 mg ofsynthetic nanoparticles has a surface area of 94 cm², larger than 6 cm².The absence of pyrogenicity of the preparation is then demonstratedeither by measuring the sum of the temperature increases in threerabbits which must not exceed 10.5, 2 or 1° C., preferentially 2.65° C.,in agreement with the European Pharmacopoeia, or by measuring thetemperature increase in each rabbit, which should not exceed 10, 5, 2, 1or 0.1° C., preferentially 0.5° C. in agreement with the AmericanPharmacopoeia, or by following the recommendations of one or morepharmacopoeias, in particular the European and American pharmacopoeias.

Preferentially, the preparation according to the invention isnon-pyrogenic or non-immunogenic. In particular, immunogenicity orpyrogenicity can be caused by non-mineral biological substances, lipids,proteins, enzymes, DNA, or RNA, whether these substances are produced bya different living organism from a humans or are synthesized at least inpart by human.

Preferentially, the preparation according to the invention isnon-pyrogenic or non-immunogenic when it comprises less than 10⁹, 10⁸,10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², 10¹, or 1 biological molecule(s), such asprotein(s), lipid(s), enzyme(s), DNA, or RNA.

In one embodiment, the preparation according to the invention can beregarded as non-pyrogenic or non-immunogenic when it does not in itselfinduce a significant response of the immune system, i.e. it does nothave a significant pharmaceutical or medical activity; in particular;the preparation does not have any anti-tumor activity in itself. As willbe understood by the person skilled in the art, the preparation can havea pharmaceutical or medical activity, such as an antitumor activity whenit is exposed to the application of a radiation, such as a magneticfield, preferentially an alternating magnetic field.

In one embodiment, the preparation, the synthetic nanoparticle, itscentral part and/or its coating can be non-pyrogenic and immunogenic orbe non-pyrogenic and non-immunogenic. Pyrogenicity may be considered asan immunogenicity caused by endotoxins.

Preferentially, the synthetic nanoparticles according to the inventionhave a low level of aggregation, possess a homogeneous distribution in agiven medium, preferentially in the preparation and/or in the organismwhere they are administered. This homogeneous distribution ischaracterized by: (i), the presence of these synthetic nanoparticles in0.1, 1, 5, 10, 25 or 50% of this medium, where this percentage can bedefined as the volume occupied by the synthetic nanoparticles divided bythe total volume of the considered medium, (ii), sedimentation of thesynthetic nanoparticles occurring in more than one hour, preferentiallyin more than one week, most preferentially in more than 15 days, (iii),a sedimentation of the synthetic nanoparticles occurring in a timelarger than the administration time of the preparation in an organism.The sedimentation of synthetic nanoparticles can be revealed by thepresence of aggregates which sediment, preferentially in a liquid, andwhich are visible to the naked eye, by optical microscope or byspectroscopic measurements, in particular by absorption.

Preferentially, the synthetic nanoparticles according to the inventionare stable in suspension or the preparation according to the inventionis stable. Stability is defined as the ability of syntheticnanoparticles to remain in suspension without sedimentation. Stabilitycan in particular be measured by absorption measurements, preferentiallyby measurements of absorption variation over time at a wavelength wherethe synthetic nanoparticles absorb, typically between 400 nm and 600 nm,most typically equal to 480 nm, at a wavelength larger than 1, 50, 100,200, 300, 400, 450, 500, 600 or 800 nm, 1, 5 or 10 μm, at a wavelengthlower than 10, 5 or 1 μm, 800, 600, 500, 450, 400, 300, 200, 100, 50 or1 nm. Preferentially, the stability is such that the optical density orabsorption of the suspension of synthetic nanoparticle does not decreaseby more than 50, 25, 10, 5, 2, 1 or 0.1%, where this percentage ofdecrease may be defined as being equal to [abs(t₀)-abs(t)]/abs(t₀),where abs(t₀) and abs(t) are the absorptions measured at the beginningand at the end of the measurement, respectively. In particular, thisdecrease can be measured over time for a period of more than 1.5, 15,25, 45 or 60 seconds, 2, 5, 10, 30, 45 or 60 minutes, 2, 5, 10, 50, 100or 1000 times the time required to administer the suspension ofsynthetic nanoparticles to an organism. It can be measured just aftermaking the preparation, a few days, a few weeks, a few months, a fewyears after making the preparation. It is preferentially measured afterhomogenization of the preparation, in particular by means of sonication,vibration, agitation.

According to one embodiment, the preparation according to the inventionis stable for an iron concentration of synthetic nanoparticles largerthan 0.01, 0.05, 1, 5, 10, 30, 50, 100, 150, 200, 250, 300, 400, 500,600, 700, 800 or 900 mg/mL, 1, 2, 5 or 10 g/mL.

In one embodiment, the synthetic nanoparticle has a homogeneity ofdistribution, a stability, which is(are) larger than that(those) of thesynthetic nanoparticles extracted from a living organism, preferentiallya magnetotactic bacterium or larger than that(those) of the central partof the synthetic nanoparticles or of SPION or of FION.

In one embodiment, the synthetic nanoparticle may have:

-   -   A common property with its central part, preferentially the        property (α), (β), (χ), (δ), (η) (see page 5-6),    -   A size, which is 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4 5, 6, 7,        8, 9, 10, 15, 20, 50, 75, 100, 250 or 500 nm, 1, 5, 10, 50 or        100 μm larger than the size of its central part and/or coating.    -   An isoelectric point, in particular an isoelectric point, which        is different from that of its central part and/or coating,    -   A zeta potential, a charge, a surface charge, in particular any        of these three parameters and/or variation as a function of pH        which is(are) different from that(those) of its central part        and/or coating.    -   A non-pyrogenicity which is lower, larger, or equal to/than that        of its central part and/or its coating.

In one embodiment, the coating according to the invention favors theattachment of substances to synthetic nanoparticles, enabling to limitthe toxicity and results in a specific organization such as: (i), achain arrangement, i.e. an arrangement of at least two syntheticnanoparticles linked together with a possible alignment ofcrystallographic axes or field lines of these nanoparticles, (ii), anarrangement in a geometric figure, (iii), ordered and characterized bythe presence of a geometric pattern, such as a circle, a rectangle, adiamond, a square, or an ellipse. Such an organization can behighlighted by TEM measurements, in particular by depositing a drop ofthe preparation of synthetic nanoparticles at a suitable concentrationto be able to observe the arrangements of the synthetic nanoparticles intheir assemblies.

In one embodiment, the coating does not originate from the organismwhich synthesizes the central part of the synthetic nanoparticles, whichmay facilitate its characterization, in particular when the structure orcomposition of the coating is known, simple, identifiable or easilycharacterizable.

In one embodiment, the coating is a non-pyrogenic substance.

In one embodiment, the coating comprises at least one compound, which isable to establish weak interactions or covalent bonds with the centralpart of the synthetic nanoparticles, in particular iron oxide.

In one embodiment, the coating comprises at least one compound able tobe chemisorbed or physisorbed on the central part of the syntheticnanoparticle.

In one embodiment, the coating comprises at least one compound able toestablish interactions or bonds with Fe²⁺ or Fe³⁺ ions, hydroxyls OH⁻,oxides O²⁻, crystalline defects of the central part, which may be in orat the surface of the central part of synthetic nanoparticles.

In one embodiment, the coating comprises at least one compound, atom,ion, or chemical function such as an acid, carboxylic acid, phosphoricacid, or sulfonic acid function, wherein the compound, atom or ioncomprised in the coating is able to establish interactions or bonds withthe central part or with at least one atom of the central part, achemical function of the central part, an ion of the central part suchas Fe²⁺, Fe³⁺, Hydroxyl OH⁻, oxide O²⁻ or a crystalline defect of thecentral part.

The atom, the chemical function or the ion of the central part may be inor at the surface of the central part of the synthetic nanoparticles.

In one embodiment, the coating is chosen from the compounds which yieldbetter heating properties for the synthetic nanoparticles than for SPIONor FION. It is thus preferred that the coating comprises substances,which are good thermal conductors.

In one embodiment, the coating is chosen from compounds which produceamong synthetic nanoparticles an organization or assembly properties,which favor the effects of a radiation or of a magnetic field such as analternating magnetic field on these synthetic nanoparticles. The effectsof a radiation or of a magnetic field may in particular be movements,vibrations, rotations, or translations of these nanoparticles.

In one embodiment, the coating has a thickness which is less than theaverage diameter of the central part of the synthetic nanoparticles,less than half, a quarter of that diameter, less than 10.5, 2.5 or 1 μm,500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2,1 or 0.5 nm. Such a thickness may in particular enable to limit thetoxicity.

In one embodiment, the coating has a thickness typically larger than theaverage diameter of the central part of the synthetic nanoparticles,larger than one-half, one-fourth of this diameter, larger than 0.1, 0.5,1, 2, 4, 8, 10, 15, 20 or 25 nm. Such a thickness may in particularenable to link the synthetic nanoparticles together or prevent theformation of aggregates. Advantageously, the presence of a sufficientlythick coating enables to prevent the synthetic nanoparticles fromsticking together, in particular under the effect of the magnetic forceswhich they exert on each other and whose intensity is the highest whenthese synthetic nanoparticles become the closest to each other.

In one embodiment, the percentage of variation of the thickness of thecoating is defined as being the smallest divided by the largestthickness of the coating that can be measured on the syntheticnanoparticle(s) in the preparation, using in particular TEM. In a firstcase, the thickness of the coating is inhomogeneous. The percentage ofvariation in thickness is then preferably lower than 10⁶, 10⁵, 10⁴, 10³,500, 100, 50, 10, 5, 1 or 0.1%. An inhomogeneous thickness may inparticular result in a distribution of magnetic forces of variableintensity. In a second case, the thickness of the coating ishomogeneous. The percentage of variation in thickness is then largerthan 0.1, 1, 5, 10, 50, 100, 500, 10³, 10⁴, 10⁵ or 10⁶%. A homogeneousthickness may in particular allow a homogeneous distribution of thesynthetic nanoparticles.

In one embodiment, the coating has an iron content lower than or equalto 1, 2, 5, 10, 10², 10³, 10⁴, 10⁵ or 10⁶ times that of the central partof the synthetic nanoparticles.

In another embodiment, the coating possesses a content in at least oneother atom than iron and oxygen which is larger than or equal to 1, 2,5, 10, 10², 10³, 10⁴, 10⁵ or 10⁶ times that of the central part ofsynthetic nanoparticles.

In one embodiment, the coating is a surfactant inducing a variation insurface tension at the surface of the synthetic nanoparticles, largerthan 10⁻⁴, 10⁻³, 10⁻², 10⁻¹, 1, 5, 10, 20, 50 or 100% where thispercentage is defined as equal to TS(CP)-TS(SN)/TS(CP), where TS(CP) andTS(SN) are surface tensions of the central parts and syntheticnanoparticles, respectively.

In one embodiment, the coating comprises carbonaceous compounds.

In one embodiment, the coating comprises at least one compound selectedfrom the group consisting of a chelator, an amphiphatic molecule, apolarized or charged polymer, a metal or silicon oxide, a metal orsilicon hydroxide, an acid, an acidic, basic, oxidized, reduced,neutral, positively charged, negatively charged, derivative of thesecompounds, and a combination of several of these compounds orderivatives.

In one embodiment, the coating comprises at least one compound selectedfrom the group consisting of a polysaccharide, a fatty acid, aphospholipid, a polymer of amino acids, polymeric or non-polymericsilica, and an aliphatic amine polymer, of an acidic, basic, oxidized,reduced, neutral, positively charged, negatively charged derivative ofthese compounds, and a combination of several of these compounds orderivatives.

In one embodiment, the coating does not comprise phospholipids orproteins or RNA or DNA or compounds of bacterial, cellular or biologicalorigin or compound derived from a magnetotactic bacterium.

In one embodiment, the coating comprises at least one function selectedfrom the group consisting of phosphoric acids, carboxylic acids,sulfonic acids, esters, amides, ketones, alcohols, phenols, thiols,amines, ethers, sulfides, acid anhydrides, acyl halides, amidines,nitriles, hydroperoxides, imines, aldehydes, peroxides, of an acidic,basic, oxidized, reduced, neutral, positively charged, negativelycharged derivative of these compounds, and a combination of several ofthese compounds or their derivatives.

In one embodiment, the coating according to the invention is chosen fromsterilizable substances, preferentially by autoclaving, biocompatible,biodegradable substances, which do not induce metabolic, immunological,cytotoxic, pharmacological effect, which can be administered by anintravenous and/or immunological route. Such a substance can bepovidone, PEG 400, poloxamer 188, dextran, phosphatidylcholine,dipalmitoyl-sn-glycero-3-phosphatidylcholine, or a derivative of thesesubstances.

The type of coating can be selected according to the followingparameters:

-   (i) Administration route of synthetic nanoparticles: for example,    for an intravenous administration, a coating that enables to prevent    macrophages from capturing synthetic nanoparticles, such as PEG or    dextran can eventually be chosen,-   (ii) Cellular internalization: to support it, a coating with a    positive charge such as poly-L-lysine can “eventually” be chosen.

In one embodiment, the preparation according to the invention can beused as drug or as diagnostic agent, in particular in the context of thetreatment of a tumor, for example using magnetic hyperthermia.

In one embodiment, a medical, veterinary or cosmetic intervention oroperation using this preparation involves at least one of the followingsequences, preferentially in the indicated chronological order:

-   (i) administration of the preparation to an organism, in particular    by a local route, muco-cutaneous, enteral, parenteral,    intra-tumoral, intravenous or intra-arterial,-   (ii) targeting of synthetic nanoparticles towards part of an    organism, such as an organ, a tumor, a blood vessel,-   (iii) treatment or detection of that part of the organism.

In one embodiment, the preparation according to the invention can beused for cosmetic applications.

In one embodiment, the preparation according to the invention isadministered at a concentration, which is larger than the maximumconcentration at which a suspension comprising chains of magnetosomeextracted from magnetotactic bacteria can be administered,preferentially 14 mg/mL in iron. It may also be administered at an ironconcentration, which is larger than or equal to 0.01, 0.05, 1, 5, 10 or15 mg/mL, preferentially 25, 50, 100 or 150 mg/mL, most preferentiallylarger than or equal to 200, 300, 400, 500, 600, 700, 800 or 900 mg/mL,1, 2, 5 or 10 g/mL. This may be enabled by the larger solubility or thelower sedimentation of the synthetic nanoparticles compared to that ofthe chains of magnetosomes extracted from magnetotactic bacteria and/orthat of SPION and/or that of FION.

In one embodiment, the preparation according to the invention can beadministered at an iron concentration lower than or equal to 1 kg/mL,500, 250, 100, 50, 10, 5, 2 or 1 g/mL, preferentially lower than orequal to 900, 700, 500 or 400 mg/mL, most preferentially lower than orequal to 300, 200, 150, 100, 10, 1 or 0.1 mg/mL.

In one embodiment, the treatment can be carried out by applying analternating magnetic field, a technique commonly referred to as magnetichyperthermia, whereas the detection can in particular be carried out bymagnetic resonance imaging (MRI).

In one embodiment, the invention relates to a pharmaceutical compositionor drug comprising, as active principle, a preparation as describedabove and optionally at least one pharmaceutically acceptable carrier.

In one embodiment, the invention relates to a medical device comprisingthe preparation according to the invention.

In an embodiment, the invention relates to a diagnostic compositioncomprising the preparation according to the invention.

In one embodiment, the invention relates to a cosmetic compositioncomprising, as cosmetic active principle, the preparation according tothe invention.

In one embodiment, the invention relates to a method for treating atumor in an individual or animal wherein one administers atherapeutically active quantity of the preparation according to theinvention.

In one embodiment, the invention relates to a method for the manufactureof a preparation as described above, comprising the following sequences:

-   (i), from a preparation of nanoparticles synthesized by a living    organism comprising a crystallized mineral central part composed    predominantly of iron oxide and a biological surrounding coating,    isolate the mineral central part;-   (ii), treat the resulting preparation to cover the central part with    a surrounding coating;-   (iii), optionally sterilize the preparation, preferentially after    sequence (i), possibly after sequence (ii).

In one embodiment, the central part of the magnetosomes is surrounded ornot by compound(s) or substance(s) not belonging to this central part.

In one embodiment, treated magnetosomes are associated withmagnetosomes, which have undergone a treatment, in particular followingthe sequence of bacterial fermentation.

In one embodiment, the sequences (i) and/or (ii) can involve:

-   (a), a chemical purification process, called the said chemical    purification process, whose objective is in particular to remove, by    a chemical method, all or part of the material surrounding the    central parts of the magnetosomes. It can consist in mixing any    suspension of the sequences (i) or (ii) with a chemical solution,    designated the said chemical solution, preferentially at a    concentration larger than 0.001, 0.01, 0.1, 1, 10 or 100 μM, 1, 10    or 100 mM, 1 M, preferentially at a concentration lower than 1000,    100, 50, 10, 5, 2 or 1 M, 500, 100, 50, 10, 5 or 1 mM, 100, 50, 10,    5, or 1 μM. This chemical solution may be a hypo-osmotic solution, a    lysis buffer, a solution for desorbing or detaching the magnetosomes    from certain surfaces, for removing all material, in particular    organic, not located in or at the surface of the magnetosome central    part, for removing some residues of detergents. This chemical    solution can comprise the following chemical substances: (i)    chemical denaturants such as sodium hydroxide, potassium hydroxide    or solution with neutral, acidic or basic pH, (ii), organic solvents    such as toluene, ether, alcohol, phenylethyl, dimethyl sulfoxide    (DMSO), benzene, methanol, chloroform, (iii), chaotropic agents such    as urea, phenol, guanidine, guanidium chloride, guandinium    thiocyanate, which are in particular able to bring hydrophobic    compounds into aqueous solutions by modifying the structure of    water, (iv), chelating agents such as ethylene diamine tetraacetic    acid (EDTA), (v), substances used in depyrogenation such as sodium    hydroxide, urea, hydrogen peroxide, enabling to remove or neutralize    endotoxins, (vi), antibiotics, such as thionines, (vii), surfactants    such as triton, Brij, Duponal, (viii), reducing agents such as    dithiothreitol (DTT), thioglycolate, β mercaptoethanol, which can in    particular enable to break bonds or disulfide bridges, (ix),    detergents, compounds with a hydrophobic hydrocarbon group and a    hydrophilic charged extremity, (x), surfactants. Among the    detergents which can be used are ionic, cationic, or anionic    detergents, nonionic detergents, zwitterionic detergents,    chaotropes, sodium dodecyl sulphate (SDS), desoxycholate, cholate,    triton, in particular triton X100, n-Dodecyl1β-D-maltoside (DDM),    digitonin, tween 20, tween 80,    3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS),    urea, guanidine hydrochloride, nonyl phenoxypolyethoxylethanol    (NP-40). The said chemical solution can also comprise water or any    of the derivative(s) of the previously mentioned chemical    substance(s);-   (b), a biological purification process, called the said biological    purification process, whose objective is in particular to remove by    a biological process all or part of the material surrounding the    central part of the magnetosomes. It can in particular involve a    living organism or a substance derived from such an organism, such    as an enzyme, in particular a lytic enzyme, a phage, a protein such    as protease or mannase;-   (c), a chemical coating process, called the chemical coating    process, consisting in mixing the suspension comprising the central    parts of the magnetosomes with any substance or combination of    substances used for coating the central part of the magnetosomes,    wherein the said substance(s) is(are) called the said coating    substance(s);-   (d), a thermal process, called the said thermal process, consisting    in exposing any suspension obtained during sequences (i) or (ii) to    a temperature gradient, in particular larger than or equal to 10,    25, 50, 100, 150, 200, 300, 500, 1000, 2000, 3000 or 5000° C. and/or    in bringing any suspension obtained during sequences (i) and/or (ii)    at a temperature, which is preferentially lower than or equal to    100, 50, 10, 0, −10, −40, −70, −77, −150, −250° C., 50, 30, 10 or 1    K, which is preferentially larger than or equal to 1, 5, 25, −250,    −70, −77, −50, −20, 0, 5, 20, 40, 100, 200, 500, 1000, 2000, 4000 or    5000° C.-   (e), a mechanical process, called the said mechanical process,    consisting in applying to any of the suspensions obtained during    sequences (i) and/or (ii): (1), a pressure, preferentially larger    than 1, 10, 100, 500, 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ or 10⁹    atmosphere(s), preferentially lower than 10⁹, 10⁸, 10⁷, 10⁶, 10⁵,    10⁴, 10³, 500, 100, 10 or 1 atmosphere(s), in particular by using an    instrument such as a high pressure homogenizer, a French press, a    disrupting bomb or a ball mill, (2), sonication, in particular at a    power larger than 0.01, 0.1, 0.5, 1, 2, 5, 7, 10, 20, 40, 100, 500    or 1000 Watt, at a power lower than 1000, 500, 100, 40, 20, 10, 7,    5, 2, 1, 0.5, 0.1 or 0.01 Watt, wherein the length of time of    sonication is preferentially larger than 1, 2, 5, 10, 20, 30, 45 or    60 second(s), larger than 2, 5, 10, 20, 30, 45, 60 or 120 minutes,    larger than 2, 3, 4 or 5 hours, preferentially less than 60, 30, 15,    5 or 1 minute(s), less than 60, 45, 30, 15, 10 or 1 second(s), (3),    irradiation, in particular using ionizing rays;-   (f), a process of magnetic selection, called the said magnetic    selection process, consisting in isolating the central part of the    magnetosomes or the treated magnetosomes from substances with no or    low magnetism, defined as being bacterial debris, un-lysed bacteria,    dissolved iron, dissolved magnetosomes or any material comprised in    any suspension of sequences (i) or (ii), which comprises no or a low    quantity of magnetosomes, preferentially less than 10⁹, 10⁸, 10⁷,    10⁶, 10⁵, 10⁴, 10³, 10² or 10 magnetosomes per milliliter of    preparation. For this purpose, a magnetic field gradient may be    applied to any suspension of the sequence (i) or (ii), where such a    suspension is preferentially comprised in a container such as a tube    or a bottle, preferentially composed of glass or of a material that    does not adsorb the modified magnetosomes or the central parts of    the magnetosomes. This magnetic field gradient enables in particular    the migration of the magnetic substances, preferentially the central    parts of the magnetosomes or the modified magnetosomes, towards the    area where the magnetic field is the most intense such as the wall    of the container, to concentrate them in this area and thus to    isolate them. This magnetic field gradient is preferentially of    larger intensity than the earth's magnetic field strength, at 0.1,    1, 10 or 100 μT, 0.1, 1, 10 or 100 mT, 0.1, 1, 10, 50 or 100 T. It    is in particular of sufficient intensity to enable magnetic    substances, such as the central parts of magnetosomes or modified    magnetosomes, to be separated from substances with no or low    magnetism. This magnetic field gradient is preferentially of lower    strength than 100 or 50 T, most preferentially at 5 or 1 T, 100, 50,    10 or 1 mT. In particular, it may be of sufficiently low strength to    avoid attracting substances with no or low magnetism. Such magnetic    field gradient can be applied by a magnet, such as a Neodymium    magnet, an electromagnet or any instrument or material able to    generate a magnetic field whose intensity varies preferentially    spatially and/or temporally. The magnetic field gradient may be    applied for more than 1.5, 10, 30, 45 or 60 second(s), 5, 10, 30, 45    or 60 minutes, 2, 5, 10, 15 or 20 hours, 1, 2 or 5 day(s), 1, 2, 3    or 4 week(s), 2, 4 or 6 months. Following application of the    magnetic field gradient, the supernatant of the suspension, which    does not comprise or comprises in small quantity the central parts    of the magnetosomes or the modified magnetosomes, can be removed and    replaced by a substance such as a solvent, preferentially    non-pyrogenic, such as non-pyrogenic water;-   (g), another selection process, called the said other selection    process, during which the central parts of the magnetosomes or the    modified magnetosomes can be isolated from the bacterial debris, in    particular by using a tangential filtration system possessing    columns with pores whose sizes either enable bacterial debris to    pass through, but not the central parts of the magnetosomes, or    enable the central parts of the magnetosomes to pass through, but    not to the bacterial debris. This size is notably lower than 100,    50, 20, 10, 5, 1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01, 0.005 or 0.0001    μM. This size may preferentially be between 0.02 μm and 2 μm,    preferentially between 0.2 μm and 1 μm, most preferentially between    0.2 μm and 0.4 μm;

In one embodiment, sequence (i) is preceded by a sequence of culture ofa living organism, such as a magnetotactic bacterium, which synthesizesmagnetosomes.

In one embodiment, the culture sequence of the living organism iscarried out under conditions that are not fully controlled, such as theculture conditions of magnetotactic AMB-1 bacteria in bottle.

In another embodiment, the sequence of culture of the living organism iscarried out under controlled conditions such as the culture conditionsof MSR-1 magnetotactic bacteria in fermenter, which involve: (a) anacidic nutritive solution comprising iron to keep the pH of the growthmedium constant during the growth of the bacteria and, (b), air supplyduring the growth of bacteria to promote this growth while keeping theoxygen concentration of the culture medium at a low value,preferentially lower than 20 mbar, most preferentially lower than 2mbar, in order to enable magnetosome synthesis.

In one embodiment, the sequence (i) is subdivided into three sequencesthat preferentially but not necessarily follow each other in theindicated order: (i1), lysis of magnetotactic bacteria, (i2), removal ofthe material, in particular organic, which is not part of the centralpart of the magnetosomes and which could not be removed during i1, (i3),recovery and washing of the suspension comprising the central parts ofthe magnetosomes. All of these sequences can be carried out using anycombination of the said processes (a) through (g), repeated one orseveral times.

In one embodiment, the sequence (i1) can begin with the concentration ofthe magnetotactic bacteria, in particular by centrifuging at 1000-10000g and/or by using the said other selection process, preferentially usinga tangential filtration system, where the size of the holes is lowerthan the size of the magnetotactic bacteria. The concentration ofbacteria at the end of this sequence may be between 1.1 and 10⁴ timeslarger than the bacterial concentration measured at the beginning of(i1). Then, the magnetotactic bacteria, concentrated or not, can belysed. Lysis can be carried out by osmotic shock, using the saidchemical process in which the said chemical solution is preferentially alysis buffer. It can be carried out by adding a larger volume of lysisbuffer than the volume of bacteria, preferentially equal to 2 to 100times the volume of bacterial lysate, most preferentially equal to 4times the volume of bacteria. At the end of this sequence, one obtains asuspension of treated magnetosomes, extracted from the bacteria, mixedwith bacterial debris and possibly with a certain quantity of non-lysedmagnetotactic bacteria.

In another embodiment, the sequence (i2) consists in treating themagnetosome suspensions obtained at the end of (i1) to remove theremaining material, in particular organic material, preferentially usingthe said chemical solution, preferentially using detergents, such asphenol or dichloromethane, making it possible to remove the membrane orthe material, in particular organic, surrounding the treatedmagnetosomes.

In another embodiment, sequence (i3) consists in washing the suspensionobtained in (i2) several times to remove all the residues coming fromthe detergent and to obtain a suspension comprising the central parts ofthe magnetosomes.

In one embodiment, the sequence (ii) consists in covering the centralpart of the magnetosomes with a coating, preferentially non-pyrogenic.It may be carried out using the said chemical coating process, inparticular by mixing the suspension comprising the central parts of themagnetosomes obtained at the end of (i3) with a suspension comprisingthe coating. This mixture can be carried out by homogenization, bysonication, preferentially low-power sonication, of less than 500, 250,100, 50, 25, 10, 5, 2 or 1 Watt, in particular using a sonicating fingeror a sonicating bath, by stirring, by heating, by radiation and thusallowing the coating to adhere to or associate with the surface of thecentral parts of the magnetosomes. The mixture can be carried out atneutral, acidic or basic pH, preferentially at a pH enabling to promotethe chemical interactions and/or chemical reactions resulting in anassociation between the coating and the central parts of themagnetosomes. The mass of the coating used is preferentially between onethousandth and one thousand times the mass of the central parts of themagnetosomes, preferentially between one hundredth and one hundred timesthis mass, most preferentially between one tenth and ten times thismass. At the end of the sequence (ii) a suspension is obtainedcomprising the central parts of the magnetosomes covered with anon-pyrogenic coating.

In one embodiment, the central part of the magnetosomes obtained at theend of the sequence (i3) is sterilized, preferentially using a methodwhich does not denature this central part, such as autoclaving. Thesequence (ii) is then preferentially carried out in a sterileenvironment in order to obtain a non-pyrogenic preparation at the end ofthe sequence (ii).

In one embodiment, the suspension obtained at the end of sequence (ii)is sterilized, preferentially using a method such as gamma rays whichdoes not destroy the coating and the central part of the magnetosomes.

In one embodiment, the yield obtained from the suspension comprising thecoated central parts of the magnetosomes is estimated as being equal tothe quantity of iron comprised in this suspension divided by thequantity of initial iron comprised in the suspension of magnetotacticbacteria before the lysis sequence. This yield is preferentially largerthan 1, 2, 5, 10, 25, 50, 75 or 100%. This yield can be optimized bycombining several of the said processes with one another and/or byrepeating one or more of the said process(es).

EXPERIMENTAL EXAMPLES Description of the Tables

Tables 1 and 2: Properties of various types of suspensions comprisingBNF-Starch, whole bacteria, central parts of uncoated magnetosomes,central parts of magnetosomes coated with poly-L-lysine, chitosan,carboxy-methyl-dextran, citric acid, oleic acid, silica, folic acid,DOPC, alendronate, neridronate, PEI, Al(OH)₃, where the species ofmagnetotactic bacteria are AMB-1 and MSR-1. In table 1: Endotoxinconcentration measured in EU per milligram of iron per milliliter ofsuspension, percentage of decrease after 20 minutes of absorption,measured at 480 nm, of 1 mg of the various suspensions, variation intemperature (ΔT) and initial slope of this variation (δT/δt) for thesuspensions brought into contact with GL-261 cells treated according tocondition 3, percentage of living cells for the suspensions brought intocontact with GL-261 cells treated according to conditions 1, withoutfield (−B), and according to condition 2, with field with a maximumtemperature increase of 45° C., % living cells (+B). The percentage ofliving cells is the number of living cells treated under conditions 1 or2 divided by the number of untreated living cell number at 37° C. Forthe first study with BNF-Starch, the percentage of living cells is thenumber of living cells treated under conditions 1 or 2 divided by thenumber of untreated living cells at 45° C. In table 2: Coating thicknessin nm, isoelectric point in pH unit, hydrodynamic size in nm, zetapotential measured in mV as a function of pH, of coated or uncoatedsynthetic nanoparticles, comprised in the various suspensions,percentage of nitrogen (% N), carbon (% C), hydrogen (% H), sulfur (% S)in coated or uncoated synthetic nanoparticles.

Table 3: Binding properties of the central parts between each other fordifferent coatings.

Material and Methods

Determination of the concentration in iron of the different nanoparticlesuspensions: The nanoparticles are first dissolved by 12N hydrochloricacid and Fe²⁺ ions are oxidized to Fe³⁺ ions with hydrogen peroxide.Potassium thiocyanate is then used to complex the Fe³⁺ ions and Fe³⁺concentration is determined by measuring the absorption of the complexat 476 nm. Transmission electron microscopy (TEM): TEM is used todetermine the size, distribution in size of the different nanoparticles,nanoparticle coating thickness and the type of nanoparticleorganization. To carry out TEM studies, the different suspensions arewashed twice and re-suspended in MilliQ autoclaved water to obtain aconcentration in iron of 300 μg/ml. 5 μl of each suspension aredeposited on top of carbon grid. Grids are dried during at least twohours at room temperature and then observed under TEM (JEOL LaB6JEM-2100).

Test of Limb Amoebae Lysate (LAL): LAL tests are carried out understerile conditions using Thermo Scientific kit 88282 called “Pierce LALChromogenic Endotoxin Quantitation Kit”. 1 ml of each suspension,homogenized by sonication and washed with non-pyrogenic water, is firstheated at 70° C. for 10 minutes in a dry bath to denature any residualproteins which could distort the results of the LAL test. 25 μl of eachsuspension comprising 10 μg of iron are then introduced into the wellsof a microplate maintained at a temperature of 37° C. throughout theduration of the experiment. 25 μl of the LAL kit solution are added toinitiate the reaction. After 10 minutes of reaction, 50 μl of thechromogenic substrate are introduced into the wells for 6 minutes toenable detection of the quantity of endotoxins. Finally, 25 μl of aceticacid are added to stop the reaction. The optical density of the obtainedsuspensions is measured at 405 nm using a microplate reader. Theconcentration of endotoxins is then estimated using a standard rangesupplied with the kit. In order to verify that the LAL test does notinterfere with the nanoparticles, a recovery rate, defined as beingequal to C_(total)/C₁+C₂, is measured, where C_(total) is the endotoxinconcentration of the nanoparticle suspensions mixed with a knownquantity of Endotoxins of 0.5 EU/mL, C₁ is the concentration ofendotoxins in the different nanoparticle suspensions and C₂=0.5 EU/mL.The recovery rate estimated during the different measurements is largerthan 50%, indicating that the nanoparticles do not interfere with theLAL test.

Elementary analyzer of carbon, hydrogen, nitrogen and sulfur (CHNS):Measurements are carried out using a CHNS analyzer (Flash EA 1112Analyzer from Thermo Fischer scientific) using 10 mg of iron of eachlyophilized suspension, enabling to determine the percentage in carbon,nitrogen, hydrogen and sulfur of these suspensions.

Scattering measurements: The zeta potential and the hydrodynamic size(hydrodynamic diameter in the case of spherical objects) of the variousnanoparticles are measured using the Malvern Instruments Zetasizer NanoZS. The spherical objects are identified with the decreasing exponentialprofile of the correlation function. For the measurements, thesuspensions of nanoparticles comprise 30 μg/ml of iron and are at a pHadjusted between 2 and 12 using solutions of hydrochloric acid andsodium hydroxide.

Absorption measurements: The variation over time of the absorbance ofthe different nanoparticle suspensions is measured at 480 nm using aUviLine9400 Secomam absorption spectrophotometer.

Determination of the primary amine concentration for 1 mg in iron of thesuspension of central parts of the synthetic nanoparticle: A method forcontrolling the purity of the central part is the dosage of amines viaTNBSA (2,4,6-trinitrobenzene acid Sulfonic acid), which has theparticularity of reacting with primary amines. The first step is to add1 mg of iron of the central part to a 0.1 M sodium bicarbonate buffersolution at pH 8.5 and then TNBSA (R′—SO3H) is added, which leads to thefollowing reaction: R—NH₂+R′—SO₃H<=>H₂SO₃+R′—NH—R. This reaction iscarried out at 37° C. for 2 h and the obtained compound being colored inyellow (R′—NH—R), its amine concentration is determined by absorption at405 nm. This concentration is equivalent to that of the central part. Acalibration curve is carried out with glycine which has a primary aminefunction.

Determination of the phosphate concentration for 1 mg in iron ofsuspension of central part of the synthetic nanoparticle: The assay ofthe phosphate groups of the central part is carried out by colorimetryAmmonium molybdate ((NH₄)₂MoO₄) first reacts in the presence ofphosphate (coming from a stock solution (DOPC) for calibration or comingfrom samples to be assayed after digestion with perchloric acid at 70%for 2 h at 130° C.). The precipitate of Molybdate is yellow andunstable. Very rapidly, ascorbic acid is added which will reduce thiscomplex to give a blue colored molybdate salt, which is a stablecompound (one heats for 5 minutes at 100° C. in a dry bath to activatethis reduction) and one measures the quantity of complexed phosphate byabsorbance at 800 nm.

Cellular toxicity and temperature measurement of the variousnanoparticles exposed or not to the application of an alternatingmagnetic field: The GL261 cells are seeded in a T175 flask untilreaching 70-80% confluence, the supernatant is removed, 4 ml ofTrypsin-EDTA at 0.25% are added to the cells, the cells are incubatedfor 5 minutes and then detached. Trypsin is deactivated by adding 30 mlof cell medium. The cells are then diluted to a concentration of 1.2510⁶ cells per milliliter after centrifugation at 700 rpm for 10 minutesat 4° C. 400 μl of cells are introduced into Eppendorf tubes in order toreach ˜5.10⁺⁵ cells per condition. The various suspensions of syntheticnanoparticles were added to the tubes at a final concentration of 1mg/ml in iron. The eppendorf tubes are then heated for 10 minutes at 37°C. 3 conditions follow. For treatment condition 1, the tubes aremaintained at 37° C. for 30 minutes using a dry heating bath. Fortreatment condition 2, the tubes are maintained at 45° C. for 30 minutesby applying an alternating magnetic field of frequency 198 kHz andaverage strength adjusted between 23 and 46 mT to maintain thetemperature at 45° C. For the treatment condition 3, the tubes areexposed to the application of an alternating magnetic field of frequency198 kHz and average strength of 32 mT for 30 minutes. Temperaturevariations over time are measured using a thermocouple probe placed inthe eppendorf tubes. After the treatments, the contents of the tubes areintroduced into a flask T 25 to which is added 6 ml of RPMI medium and10% of fetal calf serum. The cells are incubated for 24 hours in thepresence of 5% CO₂. 24 hours later, a cell viability test is carried outwith Trypan blue, enabling to discriminate the colorless living cellsfrom the colored dead cells.

Example 1 Characterization of Suspensions Comprising BNF-Starch

Nanoparticles synthesized chemically by the company Micromod, calledBNF-Starch (Reference: 10-00-102), are tested. These iron oxidenanoparticles are surrounded by hydroxyethyl starch and have ahydrodynamic diameter of 119 nm. They have an isoelectric point of pH9.5, a zeta potential which varies from 7 mV at pH 2 to −20 mV at pH 12and a percentage of carbon measured with the CHNS of 8.7%. The TEMmeasurements enable to estimate the thickness of the coating, from 1 to4 nm. The variation in absorption with time, measured at 480 nm, of asuspension comprising 1 mg of BNF-Starch does not decrease in 20minutes, indicating the stability of this suspension. An LAL test,performed on these suspensions, revealed a low level of endotoxins (<50EU/mg/ml). For a first series of measurements, table 1 shows that when amixture of a suspension of BNF-Starch and GL-261 cells is subjected tocondition 3 of the treatment, the temperature of the mixture slightlyincreases by 6.2° C. from 36.5° C. before application of the field to42.7° C. after 30 minutes of application of the field. The initial slopeof the temperature variation is estimated to be 0.009° C./sec. When thissame mixture is subjected to the treatment condition 2, table 1 showsthat the percentage of living cells is 78±5%, similar to that of 71±5%obtained in treatment condition 1, without field. This indicates the lowcytotoxicity induced by BNF-Starch on GL261 cells in the presence oftreatment condition 2 by comparison with cells heated to 45° C. In asecond series of measurements, when the mixture is subjected totreatment condition 2, table 1 shows that the percentage of living cellsis 31%, lower than 86% obtained during treatment condition 1, withoutfield when this percentage is estimated by comparison with the number ofliving cells at 37° C. This indicates the cytotoxicity induced byBNF-Starch on GL261 cells in the presence of treatment according tocondition 2 compared to cells heated to 37° C.

Example 2 Pyrogenic Chains of Magnetosomes Extracted from the StrainAMB-1

Preparation: Magnetospirillum AMB-1 bacteria (ATCC, strain 79024) arefirst introduced into sterile culture medium comprising the nutrientsand additives necessary for the proliferation of magnetotactic bacteriaand the production of magnetosomes (medium ATCC 1653) and the media arethen placed in an incubator at 30° C. for 7 days. After 7 days, themedia are centrifugated, the bacterial pellet is washed, themagnetotactic bacteria are concentrated by using a magnet, introducedinto a tube comprising 1 mL of TRIS 0.05 M, sonicated using a Sonicatingfinger for 2 hours at 30° C. at 0° C. and then washed 17 times withsterile Millipore® water using a magnet. A suspension comprisingpyrogenic magnetosome chains extracted from the magnetotactic bacteriais obtained.

Characterization: TEM measurements showed the presence in thesesuspensions of magnetosome chains with lengths between 50 and 800 nm,magnetosome sizes between 5 nm and 60 nm. The thickness of the coatingis 1 to 5 nm. The absorption of these suspensions comprising 1 mg ofiron, measured at 480 nm, decreases by 30% after 20 minutes, which showsthe low sedimentation of these suspensions. Light scatteringmeasurements on these chains indicate the presence of three chainpopulations, 5% with a hydrodynamic size (HS) 176 nm, 81% with HS 986nm, and 14% with HS 4363 nm. They have an isoelectric point of pH 4.2, azeta potential which varies from 20 mV at pH 2 to −38 mV at pH 12. TheCHNS analysis reveals a percentage of carbon in these chains of 13.9%.The endotoxin concentration of these suspensions, measured by the LALtest, was estimated at a high value of between 18,000 and 150,000 EU perml per mg of iron oxide. The presence of endotoxins was also suggestedby Fourier transform infrared absorption measurements using a NicoletFT-IR model 380 spectrometer. These spectra indicate the presence ofpeaks at 1250 cm⁻¹ and 1050 cm⁻¹, which can be attributed to thevibrations of phosphate groups of lipopolysaccharides and phospholipids.In a first series of measurements, when the pyrogenic magnetosome chainsextracted from AMB-1 are mixed with GL-261 cells and the mixture issubjected to treatment condition 3, the temperature of the mixtureincreases from 20.5° C., from 36° C. before application of the field to56.5° C. after 30 minutes of application of the field. The initial slopeof the temperature variation is estimated at 0.043° C./sec. Thisincrease in temperature is larger than that observed for BNF-Starch. Ina second series of measurements, when this mixture is subjected to thetreatment condition 2 in the presence of the field, table 1 shows thatthe percentage of living cells is low at 10% and lower than 55% obtainedduring the condition 1 of treatment, without field. This indicates thecytotoxicity induced by these pyrogenic magnetosomes on GL261 cells inthe presence of treatment under conditions 1 and 2 with a largercytotoxicity for condition 2 (in the presence of the field) than forcondition 1 (absence of the field).

Anti-tumor efficacy on U87-Luc tumors implanted in mouse brain: Efficacyexperiments were carried out on 7 groups of ten mice in which U87-Lucintracerebral tumors were grown with volumes between 1 and 29 mm³.During these experiments, the mice are fed according to the proceduresin force and watered at will. The general condition of the animals ismonitored daily, the mice are weighed every two days and are euthanizedwhen a reduction in their body weight larger than 15%, signs of pain, anunusual posture, are observed. The 7 different groups of mice received 8days after the injection of the U87-Luc cells at the injection site ofthe tumor cells (2.2.0): a solution of 2 μL of 0.9% NaCl (groups 1 and2); 2 μL, of a suspension of magnetosomes at 20 mg/mL in maghemite(groups 3, 4 and 5); 2 μL of a suspension of BNF-Starch at 20 mg/mL inmaghemite (groups 6 and 7). 8 days after injection of U87-Luc cells, themean tumor volumes for groups 1, 2, 3, 4, 5, 6 and 7 are 29, 10, 5, 3,25, 10 and 2 mm³, respectively. Groups of mice 2, 4, 5 and 7 are exposed3 times per week for 5 weeks to an alternating magnetic field of averagestrength 25 mT and frequency 198 kHz for 30 minutes. Histologicalstudies are carried out on the mice of group 4 to determine whether thetumor completely disappears 150 days after the administration of themagnetosome chains. The tissues studied in histology are taken fromeuthanized mice, the brains are extracted, fixed with a 4%paraformaldehyde solution, cut into transverse slices 2 mm thick,included in 3 μm thick paraffin blocks, collected on glass slides andthen stained with hematoxylin-eosin (H & E) to distinguish the healthyarea from the tumor area. Finally, the temperature is measured duringthe different treatments using an infrared camera.

In mice comprising only tumors, treated by the administration ofnanoparticles alone (magnetosome chain and BNF) or by the multipleapplications of the alternating magnetic field, the tumor volumesincrease rapidly to reach an average volume of 150 mm³ in less than 40days following administration of the tumor cells. Mean survival times ofmice belonging to groups 1, 2 and 6 were estimated as 38 days onaverage. These results suggest that neither the application of thealternating magnetic field nor the sole administration of BNF-Starch ormagnetosome chains had any significant anti-tumor effect on U87-Luctumors.

In mice treated by administration of magnetosome chains and multipleapplications of the magnetic field, a slight increase in temperature isobserved during the first three sessions of application of the field,which is similar for mice of groups 4 and 5 and which is 4° C. (firstsession), 2° C. (second session), and 0.5° C. (third session). Noincrease in temperature was observed in the following sessions ofapplication of the field among mice belonging to groups 4 and 5. For allthe other groups, no increase in temperature was observed. For micebelonging to group 5 with large tumors (˜25 mm³), a significant decreasein tumor volume during the 7 days following the administration of themagnetosome chains is observed, of 64%. Overall, the tumor volumeincreased significantly less for group 5 than for groups 1, 2, 3 and 6during the 28 days following the administration of the magnetosomechains. In addition, mice belonging to group 5 live an average of oneweek longer than those of group 1, 2 and 6 mice. For 40% of micebelonging to group 4 with small tumors (˜3 mm³) the average tumor volumedecreases during the 51 days following the administration of themagnetosome chains until the total disappearance of the tumor. Thesemice are totally healed. Indeed, these mice are still alive 143 daysafter the administration of the magnetosome chains (J143). The totalcure of these mice was confirmed by the study of histological sectionsof their brains, taken at D143, showing the absence of tumors andlesions. For mice of group 7, treated with BNF-Starch, the increase ofthe average tumor volume and the time of 45 days were similar to thoseof mice of group 1, 2 and 6. No anti-tumor effect was observed.

We can conclude that: (i) for 40% of mice with an average volume oftreated U87-Luc tumors of 3 mm³, it is possible to entirely destroythese tumors by administering 40 μg in maghemite of a suspension ofpyrogenic magnetosome chains extracted from AMB-1 in these tumors and byexposing these tumors to multiple applications of an alternatingmagnetic field with an average strength of 25 mT and a frequency of 198kHz.

Example 3 Pyrogenic Chains of Magnetosomes Extracted from Strain MSR-1

Preparation: MSR-1 bacteria are first cultured at 30° C. for 5 to 7 dayson an agar gel in the presence of iron and a low concentration of oxygen(0.5% O₂). Magnetic colonies are collected and cultured at 30° C. in thepresence of air for several days in an iron-free preculture mediumcomprising sources of carbon, nitrogen, minerals, trace elements andyeast extracts. The magnetotactic bacteria obtained from the precultureare grown in a fermenter of 50 liters at 30° C. in a medium similar tothe preculture medium. During growth the pH is maintained at 6.8-7 byadding an acidic nutrient medium comprising an iron source andcompressed air is introduced into the culture medium to promotebacterial growth while keeping the concentration in oxygen below 0.2% toallow the synthesis of the magnetosomes. MSR-1 bacteria issued from thefermentation are concentrated to an optical density, measured at 565 nm(OD_(565 nm)), of 110-120. 100 ml of this bacterial concentrate are thenmixed with 400 ml of 5M NaOH and heated at 60° C. for 1 h30 to 2 h tolyse the bacteria. The treated magnetosomes are then isolated from thebacterial debris by placing a Neodinium magnet overnight against thewall of the vessel comprising the suspension of lysed bacteria and byreplacing the supernatant comprising sodium hydroxide and bacterialdebris with 1X PBS. The suspension obtained is then sonicated for 20seconds at 10 W in the presence of 1 X PBS, placed against a Neodiniummagnet for 15 minutes, the supernatant is removed and the treatedmagnetosomes are resuspended in 1X PBS. This sequence of sonication andmagnetic separation is repeated four times. Pyrogenic chains ofmagnetosomes extracted from the MSR-1 strain are thus obtained.

Characterization: The TEM measurements showed the presence in thesesuspensions of magnetosome chains with lengths between 200 and 1500 nm,magnetosome sizes between 20 nm and 60 nm. The absorption of thesuspensions comprising 1 mg of iron of these chains, measured at 480 nm,decreases by 86% after 20 minutes, which shows the low stability ofthese suspensions. Light scattering measurements carried out on thesechains indicate the presence of two populations of chains withhydrodynamic sizes 535 nm and 2822 nm. They have an isoelectric point ofpH 6.4, a zeta potential which decreases from 15 mV at pH 2 to −31 mV atpH 12. The CHNS analysis reveals a carbon percentage in these chains of4.1% deduced from the first measure and 12.2% on average deduced overthe following 9 measures. In a second series of measurements, theendotoxin concentration of these suspensions, as measured by the LALtest, was estimated to be between 2000 and 17 000 UE per ml per mg ofiron. The presence of endotoxins has also been suggested by Fouriertransform infrared absorption measurements using a Nicolet FT-IR model380 spectrometer. These spectra indicate the presence of peaks at 1150cm⁻¹ and 1030 cm⁻¹, which can be attributed to the vibrations ofphosphate groups of lipopolysaccharides and phospholipids. In a firstseries of measurements, when pyrogenic magnetosome chains extracted fromMSR-1 are mixed with GL-261 cells and subjected to treatment condition3, the temperature of the mixture increases by 9.4° C., from 36.2° C.before field application to 45.6° C. after 30 minutes of application ofthe field. The initial slope of the temperature variation is estimatedat 0.012° C./sec during first measurement and at 0.019° C./sec duringsecond measurement. This increase in temperature is greater than thatobserved with BNF-Starch. When this mixture is subjected to treatmentcondition 2, table 1 shows that the percentage of living cells is low at5% in the first measurement and 12% in the second measurement and islower than 39% obtained in condition 1 of treatment during first andsecond measurements, without field. This indicates the cytotoxicityinduced by these pyrogenic magnetosomes on GL261 cells in the presenceof treatment conditions 1 and 2 with a greater cytotoxicity forcondition 2 (in the presence of the field) than for condition 1 (absenceof the field).

Example 4 Central Parts of the Magnetosomes Derived from MSR-1

Preparation: 100 μl of the suspension comprising pyrogenic magnetosomechains extracted from the MSR-1 strain obtained in example 3 are mixedwith 200 ml of a solution comprising 1% Triton X-100 and 1% SDS, themixture is heated overnight at 50° C., placed against a Neodiniummagnet, the supernatant is removed and replaced with 80 ml of phenol atpH 8. The obtained suspension is heated for 2 hours under sonication at60° C., maintained overnight at 60° C. without sonication, placedagainst a magnet, the supernatant of the suspension was removed andreplaced with 80 ml of chloroform. The suspension comprising thechloroform is placed against a Neodinium magnet, the supernatant isremoved and the residual chloroform adsorbed at the surface of thetreated magnetosomes is removed by heating these magnetosomes for 2hours under a hood. Finally, the obtained central parts of themagnetosomes are desorbed from the glass wall of the tubes whichcomprise them by adding 80 ml of 1M NaOH heated for 1 hour at 60° C. inthe sonicating bath. The suspension comprising the central parts of themagnetosomes is placed against a Neodinium magnet, the supernatant isremoved and replaced with sterile MilliQ water, the suspension issonicated for 20 seconds at 10 W. This washing sequence is repeated fourtimes. The suspension comprising the central parts of the magnetosomesis degassed with nitrogen to avoid oxidation, sterilized by autoclavingand stored at −80 degrees.

Characterizations: TEM measurements reveal the absence of coatingsurrounding the central parts of the magnetosomes. The absorption of thesuspensions comprising 1 mg of the central part of the magnetosomes,measured at 480 nm, decreases by 60 to 80% after 20 minutes, which showsthe low stability of these suspensions. The endotoxin concentration ofthese suspensions was estimated to be between 10 and 100 EU permilliliter per mg of iron oxide, which shows the pyrogenic nature ofthis suspension. The isoelectric point of these suspensions was measuredat an acid pH of 3.5. Moreover, between pH 4 and pH 6, we observe anincrease in the zeta potential from −8 mV to −1.5 mV, and between pH 6and pH 8, we observe a decrease in zeta potential from −1.5 mV to −27mV, which appears to be characteristic of the presence of aggregates.The light scattering measurements using the zetasizer reveal thepresence of 79% aggregates of hydrodynamic spherical size 3076 nm and21% aggregates of hydrodynamic spherical size 677 nm. CHNS analysis ofthese suspensions revealed carbon concentrations of 3.3%, nitrogen of0.2%. These concentrations are lower than those measured for pyrogenicmagnetosome chains extracted from MSR-1(% N=0.7 and % C=4.1) and forlyophilized whole MSR-1 bacteria (% N=11 and % C=49). These resultssuggest that the quantity of organic material surrounding the centralpart of the magnetosomes is significantly lower in the sample comprisingthe central parts of the magnetosomes than in those comprising the wholeMSR-1 bacteria or pyrogenic magnetosome chains extracted from MSR-1. Theamine assay indicates that there are between 260 ng and 1.2 μg of aminefunctions in or at the surface of the central parts in the suspensioncomprising 1 mg in iron of the central parts. The phosphate assayindicates that there are 10 μg of phosphate function in or at thesurface of the central part in the suspension comprising 1 mg in ironparts of the central part. In a second series of measurements, when thecentral parts of the magnetosomes are mixed with GL-261 cells andsubjected to the treatment condition 3, the temperature of the mixtureincreases by 9.8° C. after 30 minutes of field application. The initialslope of the temperature variation is estimated as 0.012° C./sec. Whenthis mixture is subjected to the treatment condition 2, table 1 showsthat the percentage of living cells is low at 0-9% and lower than 77%obtained in treatment condition1, without field.

Example 5 Central Parts of Magnetosomes Derived from AMB-1

Preparation: The culture of AMB-1 magnetotactic bacteria is carried outaccording to the process described in Example 3. After 7 days ofculture, the bacteria are concentrated at 25° C. using tangentialfiltration (500 kDa column, 800 rpm) in a volume of 1 liter. They arethen mixed with stirring in a solution comprising 1 mM EDTA and 30 μg/mLprotamine at pH=7.4, the liquid medium is separated from the bacteria bytangential filtration, and then the bacteria obtained are mixed with asolution of PBS at pH=7.4, the liquid medium is again separated from thebacteria by tangential filtration. The obtained suspension of bacteriais then diluted in PBS to an optical density, measured at 565 nm, whichallows lysing of the bacteria. The obtained suspension is sonicated for30 minutes at 70 W at 0° C. using a titanium sonication probe having adiameter of 13 mm. The suspension thus obtained is washed a first timeby placing a magnet against the wall of the container comprising thesuspension, by removing the supernatant, by replacing it with a solutioncomprising 10 mM HEPES and 200 mM NaCl at 6° C. and by sonicating byseries of 3 pulses at 30W for 2 seconds. It was washed 4 more timesusing a similar method, replacing the mixture of HEPES and NaCl withsterile non-pyrogenic water. At the end of the washings, suspensionscomprising magnetosome chains are obtained, as verified by TEM. Themagnetosome chains are isolated from the supernatant by using a magnet,the supernatant is replaced by a volume of 30 ml of TRI REAGENT (Sigma,reference: T9424), the resulting solution is sonicated for 2 hours at50° C. in an ultrasonic heating bath. The obtained suspension is thenplaced against a magnet at 4° C. for 1 hour, the supernatant is removedand replaced with a solution of sodium ethanoate and the mixture issonicated for 30 minutes at 37° C. with an ultrasonic sonic heatingbath. The treated magnetosomes are then washed a first time by isolatingthe supernatant with a magnet, by removing the supernatant, by replacingit with a solution of sodium ethanoate and then by sonicating thesuspension for 30 min at 37° C. in an ultrasonic heating bath. Thiswashing step is repeated 3 times in the same manner. In particular, toremove the lipid bilayer, the suspension is placed against a magnet, thesupernatant is removed and replaced with a solution comprising 1% TritonX114, 1% deoxycholate and 4 mM EDTA. The suspension is then stirredmechanically for 1 hour at 4° C., placed against a magnet, thesupernatant is removed and replaced with the solution comprising 1%Triton X114, 1% deoxycholate and 4 mM EDTA. The mixture is stirred forone hour at 37 degrees. The suspension is then washed a first time byplacing a magnet against the wall of the vessel comprising thesuspension, by removing the supernatant, by replacing it with amethanol/phosphate buffer (v/v, 1/1) mixture. Then, the obtainedsuspension is washed by being placed against a magnet, by removing thesupernatant, by replacing it with a mixture comprising methanol and aphosphate buffer and by then sonicating the obtained suspension for 5minutes at 30 W using a sonicating finger. A second wash is carried outin the same manner. The suspension is washed again by placing thesuspension against a magnet, by removing the supernatant, by replacingit with sterile non-pyrogenic water and by sonicating with a sonicatingbath at 37° C. for 1 hour. 5 additional washings with water are carriedout in the same manner. The obtained suspension is then sterilized byautoclaving at 121° C. to obtain suspensions comprising the centralparts of the magnetosomes derived from AMB-1.

Characterization: TEM reveals the absence of coating surrounding thecentral parts of the magnetosomes. CHNS analysis of these suspensionsrevealed a carbon percentage of 4.9% which was significantly lower thanthat of the entire bacterium, which was 32%, and pyrogenic magnetosomechains (13.9%). The level of endotoxins in these central parts is 20 to100 EU/mg/mL. The absorption of the suspensions comprising 1 mg of thecentral part of the magnetosomes, measured at 480 nm, decreases by 80 to90% after 20 minutes, which shows the low stability of thesesuspensions. The zeta potential of these suspensions decreases overallfrom 38 mV at pH 2 to −60 mV at pH 12. The isoelectric point of thesesuspensions was measured at an acid pH of 4.9. In a second series ofmeasurements, when the central parts of the magnetosomes are mixed withGL-261 cells and subjected to the treatment condition 3, the temperatureof the mixture increases by 23.8° C. after 30 minutes of fieldapplication. The initial slope of the temperature variation is estimatedas 0.024° C./sec. When this mixture is subjected to treatment conditions2 and 1, the percentage of living cells is 48% and 30%, respectively.

Example 6 Central Parts of Magnetosomes Derived from MSR-1, Coated withPoly-L-Lysine

Preparation: The central parts of the magnetosomes derived from MSR-1described in Example 4 are coated with poly-L-lysine under a laminarflow hood under sterile conditions. The poly (L-lysine hydrobromide)solution of molecular weight 21000 g/mol (Gmac, CAS: 25988-63-0)comprises 40 mg/ml of poly(L-lysine hydrobromide) prepared innon-pyrogenic water and filtered with a PES (polyethersulfone) filter of0.45 μm, stored at −80° C. During the coating sequence, the mass ofpoly-L-lysine used is seven times greater than the mass of the centralparts of the magnetosomes. 25 mL of a suspension comprising the centralparts of the magnetosomes at an iron concentration of 3 mg/mL areintroduced into a glass tube, the tube is positioned against a 1.3 TNdFeB magnet, the supernatant is removed, 25 ml of a suspension ofpoly(L-lysine hydrobromide) are then introduced into the tube at a finalconcentration of 20 mg/ml. The obtained suspension is then sonicated for6 minutes at 4° C. using the sonicating finger at 10° C. The tube isstirred for 24 hours at 25° C. on a wheel at a speed of 13 rotations perminute at a temperature between 4 and 8° C., the suspension is sonicatedusing the sonicating finger for 10 seconds at 10 W. To carry out a firstwash, the tube is then placed against the same magnet, the supernatantis removed and replaced by sterile MilliQ water. The suspensions arewashed in this manner between 1 and 4 times. Finally, the obtainedsuspension is sonicated for 2 minutes at 24 W in ice at a temperaturebelow 4° C. to avoid heating and the pH is adjusted to 6.8-7.2 withfiltered KOH. A suspension comprising the central parts of themagnetosomes coated with poly-L-lysine is thus obtained.

Characterization: Absorption of the suspension comprising 1 mg of thecentral parts of the magnetosomes coated with poly-L-lysine, measured at480 nm, decreased by approximately 50% after 20 minutes, indicating thelarger stability of this suspension compared with the suspensioncomprising the central parts of the uncoated magnetosomes. TEM imagesshowed the presence of a poly-L-lysine coating around the central partsof these magnetosomes with a thickness of 4 to 16 nm, an averagethickness of 8 nm and an arrangement in chains in part of thesesynthetic nanoparticles. An LAL assay carried out on these suspensionsrevealed a low endotoxin concentration of 78 EU per milliliter per mg ofiron (recovery rate of 119%). The light scattering measurements carriedout using the zetasizer reveal the presence of 92% aggregates ofhydrodynamic spherical size 2489 nm and 8% spherical object having ahydrodynamic diameter of 137 nm which may correspond to the hydrodynamicdiameter of the central parts of magnetosomes coated with poly-L-lysine.CHNS analysis revealed that the sample comprising 10 mg of the centralparts of the poly-L-lysine coated magnetosomes comprised percentages ofnitrogen of 0.4%, carbon of 3.6%, hydrogen of 0.6%, sulfur of 0.03%. Thesample comprising 10 mg of lyophilized poly-L-lysine comprises 13%nitrogen, 33% carbon, 3.2% hydrogen, 0.03% sulfur. These resultsindicate a lower concentration of carbon in the suspensions comprisingthe central parts of magnetosomes, coated or not with poly-L-lysine,than in those comprising whole bacteria or pyrogenic magnetosome chainsextracted from MSR-1. This suggests the presence of less organicmaterial in the suspensions comprising the coated or uncoatedmagnetosome central parts than in those comprising the whole bacteria orpyrogenic magnetosome chains extracted from MSR-1. The presence of thepoly-L-lysine coating around the central parts of the magnetosomes issuggested by the lower percentage of carbon in the sample comprising thecentral parts of the uncoated magnetosomes (% C=3.3%) than in thatcomprising the central parts of the magnetosomes coated withpoly-L-lysine (% C=3.6%). When 1.5 mg of a suspension comprising thecentral parts of the magnetosomes coated with poly-L-lysine are mixedwith 100 μl of 1% agar and the obtained mixture is subjected to theapplication of an alternating magnetic field of average strength 32 mTand frequency 198 kHz, the mixture reaches a heating temperature of 43°C. after 175 seconds. Gels comprising 1.5 mg of chemical nanoparticles(Micromod BNF-Starch, reference 10-00-102, and Micromod M-PEI, reference17-00-152), mixed with 100 μl of 1% agar are subjected to the samealternating magnetic field. They reach a temperature of 43° C. after 600seconds, which shows better heating properties for the suspensionscomprising the central parts of magnetosomes coated with poly-L-lysinethan for those comprising chemical nanoparticles BNF-Starch and M-PEI.When the central parts of magnetosomes coated with poly-L-lysine aremixed with GL-261 cells and the mixture is subjected to condition 3, thetemperature of the mixture increases by 11.5° C., from 37° C. beforeapplication of the field to 48.5° C. after 30 minutes of application ofthe field. The initial slope of the temperature variation is estimatedas 0.024° C./sec. When the same mixture is subjected to treatmentcondition 2, the percentage of living cells is 53% whereas it is only23% without application of the field (treatment condition 1). Thisindicates the cytotoxicity induced by the suspensions comprising thecentral parts of magnetosomes coated with poly-L-lysine on the GL261cells in the presence of the magnetic field.

Cytotoxicity: An MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium)cytotoxicity test according to ISO 10993-5, is carried out by anapproved organism under sterile conditions on L929 mouse fibroblastsusing suspensions comprising the central parts of the magnetosomescoated with poly-L-lysine at concentrations of 0.01 mg/mL, 0.1 mg/mL,0.5 mg/mL, and 1 mg/ml in iron comprised in EMEM10 medium. Thesesuspensions are first incubated with L929 cells at 37° C. in thepresence of 5% CO 2 for 24 to 26 hours. After incubation, 20 μL of astaining solution comprising MTS and the phenazine methosulfate (PMS)agent are added to the cells. The cells are then incubated again for 120to 135 minutes at 37° C. in the presence of 5% CO₂. The staining,resulting in an absorption at 492 nm, enables to demonstrate theviability of the cells. An observation of the cells under a microscopemakes it possible to confirm or invalidate the viability of the cells.The results obtained show a percentage of living cells of 100%, 92% and89% for the suspensions comprising the central parts of magnetosomescoated with poly-L-lysine at concentrations of 0.5 mg/mL, 0.1 mg/mL, and0.01 mg/mL in iron, this indicates the absence of cytotoxicity of thesesuspensions at these concentrations. For a concentration of 1 mg/ml, itwas not possible to conclude.

Pyrogenicity: In order to confirm the absence of pyrogenicity of thesuspension comprising the central parts of the poly-L-lysine-coatedmagnetosomes, suggested by the results of the LAL test, a pyrogenic testwas carried out on a rabbit, according to ISO 10993-11, by an approvedbody. To this end, the suspension comprising the central parts ofmagnetosomes coated with poly-L-lysine at an iron concentration of 5mg/ml was placed in the ultrasonic bath for 2 minutes, 1 ml of thissuspension was diluted in 119 ml of NaCl 0.9%. The temperature of thesuspension was maintained at 37° C. for 30 minutes, the suspension washomogenized and administered to three rabbits at a dose of 10 ml/kgintravenously. The body temperature of the three rabbits was measuredevery 30 minutes for 3 hours. It increases by 0.02° C. (first rabbit),0° C. (second rabbit) and 0.22° C. (third rabbit). None of the threerabbits shows a temperature increase larger than 0.5° C. and the sum ofthe temperature increases in the three rabbits, which is 0.24° C., isless than 2.65° C. The product tested is therefore not pyrogenicaccording to the criteria of the European and American pharmacopoeias.

Acute Toxicity: Acute systemic toxicity tests are performed among6-week-old C57BL/6 female mice by administering 100 μl of suspensionscomprising the central parts of the poly-L-lysine coated magnetosomes ata concentration of 0 mg, 0.5 mg, 1 mg, 2 mg, 4 mg and 8 mg in iron inthe tail of mice. The body weight of each mouse, measured daily during12 days after injection, remains stable, indicating that the maximumdose tolerated by mice is larger than 8 mg, i.e. about 400 mg/kg.

Anti-Tumor Efficacy on GL261 Tumors Implanted Subcutaneously in Mice

Using a 1 mL 25 g syringe, a volume of 50 μl comprising 2.10⁶ cells ofmurine glioblastoma GL261 is administered subcutaneously on the leftflank between the paw and the back of female mice black 6 C57BL/J. Thetumors grow for 10 to 15 days until they reach a size between about 40and 150 mm³. When the tumors have reached this size, the mice areanesthetized with isoflurane gas and maintained at 37° C. by means ofhot plates. Using a Hamilton 250 μl syringe, 50 μl of two differentnon-pyrogenic suspensions are administered at the center of tumorscomprising: (1), 5% glucose, (2), the central parts of the magnetosomescoated with poly-L-lysine at a concentration of 50 mg/mL in iron, mixedwith 5% glucose. The suspension 2 is administered at a quantity,measured in μg of iron, equal to 20.t, where t is the size of thetreated tumors in mm³. Then, the mice are exposed (or not) for 30minutes to an alternating magnetic field of frequency 198 kHz andaverage intensity strength between 9 mT and 28 mT to maintain theintratumor temperature at a value between 43° C. and 46° C. during thefirst three field sessions. During the following sessions of applicationof the field, the average strength of the alternating magnetic field isfixed at 28 mT. The intratumor temperature is measured using athermocouple. The sessions of application of the magnetic field arerepeated 3 times per week, 15 times in total. Mice whose tumors continueto increase following two sessions of heating undergo a secondadministration of the suspension (2). Tumor sizes are measured with acaliper and the tumor volumes are estimated using the formulaV_(tumoral)=0.5 (L.1²), where L and 1 represent the length and width ofthe tumors, respectively. The mice are euthanized when the tumor volumeexceeds 1000 mm³ and/or when the weight of the mice has decreased bymore than 20% from one measurement to another. According to thisprotocol, survival and tumor volume monitoring curves are performedduring 71 days after administration of the nanoparticle suspensions.

It is observed that 5 out of 10 mice treated with the central parts ofthe poly-L-lysine coated magnetosomes and several applications of themagnetic field are totally cured 15 days after the beginning of theheating sessions. In these mice, there is no visible trace of the tumoror its sequelae (crust or scar). The remaining 5 mice treated with thecentral parts of the coated magnetosomes and application of the magneticfield, which are not totally cured, are euthanized about 30 days afterthe beginning of treatment. The mice treated with the central parts ofthe poly-L-lysine coated magnetosomes without application of themagnetic field are all euthanized about 12 days after the beginning oftreatment. The survival rates, 50 days after the beginning of treatment,are 60% for the mice treated with the suspension comprising the centralparts of the coated magnetosomes and application of the alternatingmagnetic field and 0% for the mice treated with the suspensioncomprising the coated central parts without field application. Withoutapplication of the magnetic field, all mice died less than 20 days afterinitiation of treatment.

Anti-Tumor Efficacy on U87 Tumors Implanted in Mouse Brain

The protocols for administration of the tumor cells and for monitoringthe mice are identical to those described in Example 3. 6 days afteradministration of the U87-Luc tumor cells in the mouse brain, when thetumors reach a size between 0.8 and 2 mm³, 2 μl of two differentsuspensions are administered at coordinates (0.2.2) in brains ofanesthetized mice. For Groups 1 and 2, comprising 9 mice each, the micereceived a suspension comprising the central parts of the poly-L-lysinecoated magnetosomes at an iron concentration of 250 mg/mL mixed with 5%glucose. For groups 3 and 4, comprising 9 mice each, mice were given asuspension comprising 5% glucose. Group 1 and 3 mice are not exposed toa magnetic field. Groups 2 and 4 are exposed for 30 minutes to analternating magnetic field of average strength 25 mT and frequency of198 KHz three times per week for 6 weeks. For group 2, 4 mice withtumors regrowth receive an additional treatment 8 weeks afterimplantation of the U87-Luc cells consisting in a second administrationof 2 μl of the suspension comprising the central parts of thepoly-L-lysine coated magnetosomes at an iron concentration of 147 mg/mLmixed with 5% glucose and application of the alternating magnetic fieldof average strength 25 mT and frequency of 198 KHz three times per weekfor 3 weeks. Variations in temperature are measured during the differenttreatments using an infrared camera.

In mice treated by administration of the suspensions comprising thecentral parts of the poly-L-lysine coated magnetosomes and by multipleapplications of the alternating magnetic field an increase intemperature was observed during the first sixteen field applicationsessions for the mice of group 2, which is in average 8° C. (first 3sessions), 5° C. (12 following sessions), and 1° C. (16th session). Noincrease in temperature was observed in the following sessions ofapplication of the field for the mice of group 2. For the four mice ofgroup 2 which had been further treated following the 16th heatingsessions due to the total disappearance of the tumor, a temperatureincrease of 5.5° C. is observed during the 9 additional sessions ofapplication of the field. For groups 1, 3 and 4, no increase intemperature was observed.

In mice of groups 3 and 4, the tumor volumes increase rapidly to reachan average volume of 200 mm³ in less than 45 days following theadministration of the tumor cells. The mean survival times of mice ofgroups 3 and 4 were estimated to be 40 days on average following theadministration of the tumor cells, suggesting that the only applicationof the alternating magnetic field has no anti-tumor effect. For mice ofgroup 1, the tumor volume increased by an average of 5 mm³ per week,much less than in groups 3 and 4. In addition, 13 weeks afterimplantation of U87-Luc cells, 50% of mice of group 1 are alive, havingan average tumor volume of 56 mm³. The treatment of mice of group 1 bythe sole administration of a suspension comprising the central parts ofmagnetosomes coated with poly-L-lysine enables to slower tumor growthand to improve the survival compared with mice of groups 3 and 4. Themean tumor volume decreases during the 85 days following theadministration of a suspension comprising the central parts ofmagnetosomes coated with poly-L-lysine until the total disappearance ofthe tumor which is observed 42 days following the administration ofthese suspensions (J42) for 66% of the mice. For the 4 mice in group 2who received a second treatment because of the non-disappearance of thetumors, the tumor disappeared completely 20 days after the beginning ofthe second treatment. Overall, 91 days after administration of U87-Luccells, all mice of group 2 are still alive and do not showbioluminescence signal, suggesting cure.

We can conclude that: (i), when the suspensions comprising the centralparts of the magnetosomes coated with poly-L-lysine are administered inU87-Luc tumors, tumor growth is slowed down, which could be explained bythe high concentration of or by the presence of poly-L-lysine, (ii),when these same suspensions are administered in U87-Luc tumors andsubjected to multiple applications of an alternating magnetic field of198 kHz frequency and average strength 25 mT, it is possible tocompletely eliminate these tumors.

Example 7 Central Parts of Magnetosomes Derived from MSR-1 Coated withChitosan Preparation

A suspension comprising 1 mg of the central part of the magnetosomesmixed with sterile non-pyrogenic water is sonicated for 5 minutes with asonicating finger at a power of 5 W with pulses of 0.1 second, separatedby intervals of 0.1 second. 0.25 mg of chitosan (chitosan hydrochlorideCRS, reference sigma Y0000104) is mixed with the suspension comprising 1mg of the central part of the magnetosomes and the pH of the suspensionis adjusted to 6.2 with 1M NaOH. The obtained mixture is sonicated withthe sonicating finger at a power of 5 W, with pulses of 0.1 seconds,separated by intervals of 0.1 seconds, for 60 minutes at 45° C., thenfor 15 minutes at 60° C., then for 15 minutes at 70° C. The obtainedsuspension is washed a first time by placing a Neodinium magnet againstthe wall of the glass tube which comprises the suspension, by removingthe supernatant and by replacing it with sterile non-pyrogenic water. Itis washed a second time in the same manner.

Characterization: The endotoxin level of these magnetosomes coated withchitosan is 25 EU/mg/mL. The absorption, measured at 480 nm, of asuspension comprising 1 mg of the central parts of magnetosomes coatedwith chitosan decreased by 3% during the 20 minutes of measurement,which shows the stability of this suspension. TEM measurements carriedout on the central parts of magnetosomes coated with chitosan indicatean arrangement of these synthetic nanoparticles in chain, the presenceof aggregates and a coating surrounding the central parts of themagnetosomes with an average thickness of 6 nm. When a suspensioncomprising the central parts of magnetosomes coated with chitosan ismixed with GL-261 cells and the mixture is subjected to treatmentcondition 3, the temperature of the mixture increases by 8.5° C. Theinitial slope of the temperature variation is estimated as 0.009°C./sec. during a first series of measurements. When this same mixture issubjected to treatment condition 2, the percentage of living cells is0-5% whereas it is 20% in the absence of field (treatment condition 1).This shows the destruction efficacy of tumor cells by the central partsof magnetosomes coated with chitosan in treatment condition 2. Theisoelectric point of these magnetosomes is estimated at pH 11. The zetapotential varies between 46 mV at pH 2 to −55 mV at pH 12. Thehydrodynamic sizes of these magnetosomes are estimated at 273 nm for 7%and 1908 nm for 93% of them. The CHNS analysis indicates that theircarbon percentage is 3.2%, close to the 3.3% carbon percentage measuredfor uncoated magnetosomes.

Example 8 Central Parts of the Magnetosomes Derived from MSR-1, Coatedwith Carboxy-Methyl-Dextran

Preparation: A suspension comprising 1 mg of the central part of themagnetosomes mixed with sterile non-pyrogenic water is sonicated for 5minutes with the sonicating finger at a power of 5 W with pulses of 0.1seconds, separated by intervals of 0.1 second. 4 mg ofcarboxy-methyl-dextran (reference SIGMA 86524-10G-F) are mixed with 1 mgof the central part of the magnetosomes and the pH of the mixture isadjusted to 3.5 with 1 M hydrochloric acid. The resulting mixture issonicated for 60 minutes at 45 degrees using the sonicating finger at apower of 5 W, with pulses of 0.1 seconds, separated by 0.1 secondintervals. The obtained suspension is washed three times. For the firstwash, a magnet is placed against the wall of the tube, the supernatantis removed and replaced by sterile non-pyrogenic water. The obtainedsuspension is washed a second, then a third time in the same manner.

Characterization: In a first series of measurements, the absorption,measured at 480 nm, of a suspension comprising 1 mg of the central partsof the magnetosomes coated with carboxy-methyl-dextran does not decreaseduring the 20 minutes of measurement, which shows the stability of thissuspension. The TEM measurements enable to estimate a thickness of thecoating surrounding the central parts of the magnetosomes between 2 and20 nm. The light scattering measurements of the suspensions comprisingthe central parts of the magnetosomes coated with carboxy-methyl-dextranreveal the presence of 79% spherical objects with hydrodynamic diameter1359 nm (population 1), 6% spherical objects with hydrodynamic diameter5124 nm (population 2) and 15% of spherical objects with a hydrodynamicdiameter of 331 nm (population 3). The size of the population 3 couldcorrespond to that of the central parts of the magnetosomes coated withcarboxy-methyl-dextran. The isoelectric point is estimated to be pH=3.4and the zeta potential decreases by 20 mV at pH=2 to −31 mV at pH=12.When a suspension comprising the central parts of the magnetosomescoated with carboxy-methyl-dextran is mixed with GL-261 cells and themixture is subjected to treatment condition 3, the temperature of themixture increases by 29° C. The initial slope of the temperaturevariation is estimated as 0.023° C./sec. When this same mixture issubjected to the treatment condition 2, the percentage of living cellsis 11% whereas it is 63% in the absence of field (treatment condition1). This shows the destruction efficacy of the tumor cells by thecentral parts of the magnetosomes coated with citric acid usingtreatment condition 2. The percentage of carbon measured with the CHNSin these magnetosomes is 3.7%.

Example 9 Central Parts of Magnetosomes Derived from MSR-1, Coated withCitric Acid

Preparation: A suspension comprising 50 mg of the central parts of themagnetosomes mixed with 4.5 ml of sterile non-pyrogenic water issonicated for 5 minutes with the sonicating finger at a power of 5W withpulses of 0.1 seconds separated by intervals between pulses of 0.1seconds. 20 mg of this suspension, comprised in a volume of 1.8 ml, aremixed with 35 mg of citric acid monohydrate (reference Sigma 33114-500G) comprised in 8 ml of sterile non-pyrogenic water. In thispreparation, the mass of iron is 1.75 times larger than the mass ofcitric acid. The pH of the suspension is adjusted to 6 with 1 M sodiumhydroxide, the obtained mixture is sonicated at 90° C. for one hour. Theobtained suspension is washed a first time by placing a Neodinium magnetagainst the wall of the glass tube, by removing the supernatant and byreplacing it with sterile non-pyrogenic water. It is washed a secondtime in the same manner.

Characterization: The absorption, measured at 480 nm, of a suspensioncomprising 1 mg of the central parts of the magnetosomes coated withcitric acid decreases by approximately 15% in 20 minutes, which showsthe stability of this suspension. The TEM images of these syntheticnanoparticles reveal the presence of numerous chains, a good dispersionof these synthetic nanoparticles, low aggregation and the presence of acoating around the central part of these magnetosomes with a thicknessof 1 to 15 nm. A LAL test, carried out on these suspensions, revealed alow endotoxin concentration of 19 EU per milliliter per mg of iron(recovery rate of 188%). CHNS analysis, carried out on these lyophilizedsuspensions, revealed a percentage of nitrogen of 0.8%, carbon of 3.7%,hydrogen of 0.3% and absence of sulfur. The sample comprising onlylyophilized citric acid comprises a percentage of nitrogen of 0%, carbonof 36%, hydrogen of 4.7% and no sulfur. These results indicate a lowerpercentage of carbon in the suspensions comprising the central parts ofthe magnetosomes, whether or not coated with citric acid, than in thosecomprising the whole bacteria or pyrogenic magnetosome chains extractedfrom MSR-1. This may also suggest the presence of less organic materialin suspensions comprising the central parts of the magnetosomes, whetheror not coated with citric acid, than in those comprising whole bacteriaor pyrogenic magnetosome chains extracted from MSR-1. The presence ofthe coating around the central parts of magnetosomes coated with citricacid is suggested by the lower percentage of carbon in the lyophilizedsuspension comprising the central part of the magnetosomes (% C=3.3%)than in the lyophilized suspension comprising the central parts ofmagnetosomes coated with citric acid (% C=3.7%). When a suspensioncomprising the central parts of the magnetosomes coated with citric acidis mixed with GL-261 cells and the mixture is subjected to the treatmentcondition 3, the temperature of the mixture increases by 25° C., from35° C. before application of the field to 60° C. after application ofthe field. The initial slope of the temperature variation is estimatedas 0.038° C./sec. When this same mixture is subjected to treatmentcondition 2, the percentage of living cells is 26% whereas it is 57% inthe absence of field (condition 1 of treatment). This shows thedestruction efficacy of the tumor cells by the central parts of themagnetosomes coated with citric acid in the treatment condition 2. Thelight scattering measurements of these suspensions reveal the presenceof non-spherical objects, which may correspond to chains, ofhydrodynamic size 788 nm. The isoelectric point is estimated to bepH=3.7 and the zeta potential decreases from 25 mV at pH=2 to −38 mV atpH=12.

Example 10 Central Parts of Magnetosomes Derived from MSR-1, Coated withOleic Acid, Prepared According to the First Protocol

Preparation: We add to 5 mL of a suspension comprising 5 mg of thecentral part of the magnetosomes at a concentration of 1 mg/mL of iron,10 μl of an ammonia NH₄OH solution (25% in molecular weight) to obtain apH between 10 and 11. The obtained suspension is sonicated in a waterbath at 80° C. for 15 minutes with a sonicating finger at a power of 6-7W with pulses of 0.2 seconds separated from each other by 0.5 secondspauses. Then, either 267 μl of a 211 mM oleic acid solution(condition 1) or 50 μl of a 32 mM (0.4 mg) oleic acid solution(condition 2) are added. The mixture is then sonicated with thesonicating finger for 1 hour at a power of 6-7 W, with pulses of 0.2seconds, separated by interval of pulses of 0.5 seconds. The obtainedsuspension is then washed with a Neodinium magnet which is placedagainst the glass tube comprising the suspension, the supernatant isremoved and replaced by sterile non-pyrogenic water. The suspension iswashed a second time, then a third and a fourth time in the same mannerAfter the last washing, aliquots are taken in order to carry out thevarious characterization tests. The suspension was kept at 4° C. untiluse.

Characterization: The absorption, measured at 480 nm, of a suspensioncomprising the central parts of magnetosomes coated with oleic acid doesnot decrease for 20 minutes, indicating its stability. TEM images of thecentral parts of magnetosomes coated with oleic acid prepared accordingto Condition 2 demonstrated the presence of nanoparticle aggregates anda coating surrounding the central parts of the magnetosomes with athickness of 0.5 to 5 nm. The light scattering measurements carried outon these coated magnetosomes, synthesized according to condition 2,reveal the presence of non-aggregated, stable spherical objects with ahydrodynamic diameter of 123 nm which can correspond to the hydrodynamicdiameter of these coated magnetosomes. The isoelectric point isestimated at pH=3.5 and the zeta potential shows the presence of twopopulations whose zeta potential decreases for one population from 30 mVat pH=2 to −60 mV at pH=12 and for the other population from −10 mV atpH=6 to −35 mV at pH=12. When a suspension comprising the central partsof the magnetosomes coated with oleic acid is mixed with GL-261 cellsand the mixture is subjected to treatment condition 3, the temperatureof the mixture increases by 28° C. The initial slope of the temperaturevariation is estimated as 0.051° C./sec.

Example 11 Central Parts of Magnetosomes Derived from MSR-1, Coated withOleic Acid, Prepared According to the Second Protocol

Preparation: The central part may be coated with oleic acid in thefollowing manner We added to 1 mL of a suspension comprising 10 mg ofthe central parts of the magnetosomes at a concentration of 1 mg/mL iniron 100 mg of a solution of oleic acid at 10 mg/mL at pH 11. Theobtained suspension is sonicated for 5 minutes with a sonicating fingerat a power of 20 W, continuously and at ambient temperature. Thesuspension is then frozen at −80° C. for 30 minutes and then heated at80° C. for 5 minutes. Then the mixture is sonicated with a sonicatingfinger for 1.5 hours at a power of 10 W, with pulses of 3 seconds,separated by intervals between pulses of 3 seconds. The obtainedsuspension is then washed with a Neodinium magnet which is placedagainst the glass tube comprising the suspension, the supernatant isremoved and replaced by sterile non-pyrogenic water. The suspension iswashed a second time and a third time in the same manner After the lastwashing, aliquots are taken in order to carry out the variouscharacterization tests. The suspension was kept at 4° C. until use.

Characterization: The properties of the central parts of magnetosomescoated with oleic acid may be as follows. The absorption, measured at480 nm, of a suspension of the central parts of the magnetosomes coatedwith oleic acid comprising 1 mg of iron does not decrease for 20minutes, indicating its stability. When a suspension comprising thecentral parts of magnetosomes coated with oleic acid is mixed withGL-261 cells and the mixture is subjected to treatment condition 3, thetemperature of the mixture increases by 5° C. The initial slope of thetemperature variation is estimated as 0.012° C./sec. When this mixtureis subjected to treatment condition 2, the percentage of living cells is14% whereas it is 53% in the absence of field (treatment condition 1).The percentage of carbon in these magnetosomes is 3.4%.

Example 12 Central Parts of Magnetosomes Derived from MSR-1, Coated withFolic Acid

Preparation: A suspension comprising 50 mg in iron of the central partsof the magnetosomes mixed with 4.5 mL of sterile non-pyrogenic water issonicated for 5 minutes using the sonicating finger at a power of 30 Wwith pulses of 30 seconds, intervals of 10 seconds between pulses. A 1.8mL volume, comprising 20 mg in iron of the central parts of themagnetosomes, is introduced into a 10 mL sterile glass tube placedagainst a magnet, the supernatant is removed and replaced with 8 mL of asolution at 2 mg/mL of folic acid (Fisher BioReagents, reference:59-30-3) mixed with sterile non-pyrogenic water, previously adjusted topH 9.5 with a 1 M solution of sodium hydroxide and sterilized byfiltration (filter 0.45 μm). In this preparation, the mass of iron is1.25 times larger than the mass of folic acid. The obtained mixture issonicated for 1.5 hours with the sonicating finger at a power of 30 Wwith pulses of 30 seconds, separated by intervals between pulses of 10seconds. The obtained suspension of volume 8 mL is comprised in a 10 mLglass tube. It is washed once by placing a Neodinium magnet against thewall of the glass tube, by removing the supernatant and then byreplacing it with pyclerotic HyClone water and by sonicating for 1minute at 30W. It is washed four more times in the same manner.

Characterization: The absorption, measured at 480 nm, of a suspensioncomprising 1 mg of the central parts of magnetosomes coated with folicacid does not decrease during 20 minutes, which shows the stability ofthis suspension. The diffusion measurements of this suspension show thepresence of 90% of spherical aggregates with a hydrodynamic diameter of2876 nm and 10% of spherical objects with a hydrodynamic diameter of 235nm which could correspond to the magnetosomes coated with folic acid.The isoelectric point of this suspension was estimated to be pH 7.9 andthe zeta potential decreased from 45 mV at pH 2 to −43 mV at pH 12. Thethickness of the coating surrounding the central portions of thesemagnetosomes was measured to be 1 to 4 nm. When a suspension comprisingthe central parts of the magnetosomes coated with citric acid is mixedwith GL-261 cells and the mixture is subjected to treatment condition 3,the temperature of the mixture increases by 20° C. The initial slope ofthe temperature variation is estimated as 0.042° C./sec. When this samemixture is subjected to treatment condition 2, the percentage of livingcells is 9% whereas it is 93% in the absence of field (treatmentcondition 1). This shows the destruction efficacy of the tumor cells bythe central parts of the magnetosomes coated with folic acid intreatment condition 2. The percentage of carbon in these magnetosomes isestimated at 3.9%.

Example 13 Central Parts of Magnetosomes Derived from MSR-1, Coated withDOPC

Preparation: 40 mg of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine,reference Sigma: P6354) were solubilized in 100 μL of chloroform in a 10mL glass tube and then the solvent was evaporated for 10 minutes usinginert gas (nitrogen) to form a homogeneous lipid film in the tube. Asuspension comprising the central parts of magnetosomes with 20 mg ofiron mixed with 8 ml of sterile non-pyrogenic water, is introduced intothe tube comprising the lipid film and is then sonicated for 1.5 hourswith the sonicating finger at a power of 30 W with pulses of 10 seconds,separated by intervals of 0.5 seconds. During this preparation, the massof DOPC is twice as high as the mass of iron. The obtained suspension iswashed a first time by placing a Neodinium magnet against the glass tubewall, by removing the supernatant and then by replacing it with sterilenonpyrogenic water and by sonicating for 1 minute at 30W. It is washedfive more times in the same manner.

Characterization: In a first series of measurements, the absorption,measured at 480 nm, of a suspension comprising the central parts ofmagnetosomes coated with DOPC does not decrease during the 20 minutes ofmeasurement, which shows the stability of this suspension. The TEMimages indicate the presence of a coating surrounding the central partsof these magnetosomes of thickness 0.6 to 3 nm. The light scatteringmeasurements carried out on these suspensions reveal the presence of 87%spherical aggregates with a hydrodynamic diameter of 1871 nm and 13% ofspherical objects with a hydrodynamic diameter of 278 nm which couldcorrespond to the sizes of the central parts of the magnetosomes coatedwith DOPC. The isoelectric point is measured at pH 3 and the zetapotential decreases from 10 mV at pH 2 to −35 mV at pH 12. When asuspension comprising the central parts of the magnetosomes coated withDOPC is mixed with GL-261 cells and the mixture is subjected totreatment condition 3, the temperature of the mixture increases by 33.5°C. The initial slope of the temperature variation is estimated as 0.05°C./sec. When this same mixture is subjected to treatment condition 2,the percentage of living cells is 0-5% whereas it is 13% withoutapplication of the field (treatment condition 1). This indicates thecytotoxicity induced by the central parts of the DOPC-coatedmagnetosomes on GL261 cells in the presence of an alternating magneticfield. The percentage of carbon in these magnetosomes is estimated as7.5%.

Example 14 Central Parts of Magnetosomes Derived from AMB-1, Coated withDOPC

Preparation: In a 35 mL conical glass tube, 280 mg of DOPC(1,2-dioleoyl-sn-glycero-3-phosphocholine, reference Sigma: P6354) aresolubilized in 500 μL of chloroform and then the solvent is evaporatedduring 15 minutes with nitrogen to form a homogeneous lipid film in thetube. A suspension comprising the central parts of the magnetosomes with10 mg of iron mixed with 15 ml of sterile non-pyrogenic water isintroduced into the tube comprising the lipid film and is then sonicatedfor 30 minutes with the sonicating finger at a power of 20 W with pulsesof 10 seconds, separated by intervals between pulses of 20 seconds.During this preparation, the mass of DOPC is 28 times higher than themass of iron. The resulting suspension was purified using a SEPHADEXG-25 size exclusion column (GE Healthcare, Buckinghamshire, UK). Thebrown colored suspension is collected and then concentrated by placing aNeodinium magnet against the glass tube wall, by removing a portion ofthe supernatant which is replaced by sterile non-pyrogenic water and bysonicating the sample for 1 minute at 30W.

Characterization: The TEM images of these suspensions enable to estimatecoating thickness at 50 to 150 nm.

Example 15 Central Parts of Magnetosomes Derived from AMB-1, Coated withAlendronate

Preparation: A 2 mL volume, comprising 10 mg in iron, was introducedinto a 25 mL glass tube (non-pyrogenic) and then placed against amagnet. The supernatant was removed and then replaced with 10 ml of asolution comprising 11.6 mg/ml alendronate mixed with sterile MilliQwater, previously adjusted to pH 2.5 with a 0.1 M solution ofhydrochloric acid and sterilized by Filtration (0.45 μm filter). Duringthis preparation, the mass of alendronate is 11.6 times higher than themass of iron. The obtained mixture is continuously sonicated for 15minutes at the sonicating finger with a power of 30 W. The suspension isthen heated under a microwave at 70 W, 3 times during 1 minute with a 5minutes interval, where the sample is cooled in an ice bath. Thesuspension is washed a first time by placing a Neodinium magnet againstthe glass tube wall, by removing the supernatant and then by replacingit with sterile MilliQ water and by sonicating for 1 minute at 30 W. Itis washed ten times in the same manner by placing a Neodinium magnetagainst the glass tube wall, by removing the supernatant and then byreplacing it with sterile MilliQ water and by sonicating for 1 minute at30 W.

Characterization: The measurements of the LAL test enable to estimatethe quantity of endotoxins in these suspensions as between 53 and 144EU/mg/mL. The absorption, measured at 480 nm, of a suspension of thecentral parts of magnetosomes coated with alendronate comprising 1 mg iniron decreased by 1% during the 20 minute measurement, demonstrating thestability of this suspension. TEM measurements indicate that a matrixsurrounds these magnetosomes. These magnetosomes possess an isoelectricpoint of pH 3.5, hydrodynamic sizes of 527 nm for 14% of them and 2735nm for 86% of them, a zeta potential which varies from 25 mV at pH 2 to−47 mV at pH 12. When a suspension comprising the central parts ofmagnetosomes coated with alendronate is mixed with GL-261 cells and themixture is subjected to treatment condition 3, the temperature of themixture increases by 18.3° C. The initial slope of the temperaturevariation is estimated as 0.28° C./sec. When this same mixture issubjected to treatment condition 2, the percentage of living cells is 2%whereas it is 17% without application of the field (treatment condition1). This indicates the cytotoxicity induced by the central parts ofmagnetosomes coated with alendronate on GL261 cells in the presence ofan alternating magnetic field. The percentage of carbon in thesemagnetosomes is estimated to be 9%.

Example 16 Central Parts of Magnetosomes Derived from MSR-1, Coated withNeridronate

Preparation: A suspension comprising 50 mg in iron of the central partsof the magnetosomes, mixed with 4.5 mL of sterile non-pyrogenic water,is sonicated for 5 minutes with the sonicating finger at a power of 30W, with 30 seconds pulses separated by intervals between pulses of 10seconds. A volume of 1.8 mL, comprising 20 mg in iron of the obtainedsuspension, is introduced into a 10 mL glass tube placed against amagnet, the supernatant of the suspension is removed and replaced with 8mL of a solution comprising 20 mg/mL of neridronate mixed with sterilenon-pyrogenic water and adjusted to pH 2.5. In this preparation, themass of neridronate is 8 times higher than the mass of iron. The mixtureobtained is sonicated for 2 hours using the sonicating finger at a powerof 30 W, with pulses of 30 seconds, separated by intervals of 10seconds. The suspension is washed a first time by placing a Neodiniummagnet against the wall of the glass tube, by removing the supernatantand then by replacing it with sterile non-pyrogenic water previouslyadjusted to pH 11. It is washed five more times in the same way.

Characterization: The absorption, measured at 480 nm, of the suspensioncomprising the central parts of the magnetosomes coated with neridronatedoes not decrease for 20 minutes, indicating the stability of thissuspension. The TEM measurements indicate the presence of a coatingaround the central parts of the magnetosomes with a thickness of 19 to200 nm. The light scattering measurements carried out on thesesuspensions indicate the presence of 1% of spherical aggregates with ahydrodynamic diameter of 5560 nm, of 59% of spherical aggregates with ahydrodynamic diameter of 710 nm, and of 40% of spherical objects ofhydrodynamic diameter 207 nm that may correspond to the central parts ofthe magnetosomes coated with neridronate. When a suspension comprisingthe central parts of magnetosomes coated with neridronate is mixed withGL-261 cells and subjected to the treatment condition 3, the temperatureof the mixture increases from 37.2° C. before application of the fieldto 43.9° C. after 30 minutes of field application. The initial slope ofthe temperature variation is estimated as 0.017° C./sec. When this samemixture is subjected to treatment condition 2, the percentage of livingcells is 28% whereas it is 44% without application of the field(treatment condition 1). This indicates the cytotoxicity induced by thecentral parts of the magnetosomes coated with neridronate on the GL261cells in the presence of treatment condition 2. The isoelectric point ofthese synthetic nanoparticles coated with neridronate is estimated at pH3.5 and the zeta potential decreases from 40 mV at pH 2 to −42 mV at pH12. The carbon percentage of these magnetosomes is estimated at 18.1%.

Example 17 Central Parts of Magnetosomes Derived from MSR-1, Coated withPEI

Preparation: A suspension of magnetosome minerals comprising 50 mg iniron mixed with 4.5 mL of sterile non-pyrogenic water is sonicated for 5minutes with the sonicating finger at a power of 30 W with pulses of 30seconds separated by intervals of 10 seconds. A 1.8 mL volume comprising20 mg in iron of this suspension is introduced into a 10 mL glass tubeplaced against a magnet. The supernatant is removed and replaced with 8mL of a PEI solution at concentration 25 mg/mL, mixed with sterilenon-pyrogenic water with a pH adjusted to 9.5. In this preparation, themass of PEI is 10 times larger than the mass of iron. The obtainedmixture is sonicated for 2 hours with the sonicating finger at a powerof 30 W, with pulses of 30 seconds, separated by intervals of 10seconds. The obtained suspension is washed a first time by placing aNeodinium magnet against the glass tube wall, by removing thesupernatant, by replacing it with sterile non-pyrogenic water and bysonicating for 1 minute at 30 W. It is washed five more times in thesame manner.

Characterization: The absorption of the suspension comprising thecentral parts of magnetosomes coated with the PEI, measured at 480 nm,does not decrease during the 20 minutes of measurement, which shows thestability of this suspension. The TEM measurements indicate a coatingthickness of 8 to 10 nm. The light scattering measurements of thissuspension indicate the presence of spherical objects with a 175 nmhydrodynamic diameter which could correspond to the central parts of themagnetosomes coated with PEI. The isoelectric point is estimated at 11and the zeta potential decreases from 42 mV at pH 2 to −16 mV at pH 12.The CHNS measurements show a nitrogen percentage of 1.1% and a carbonpercentage of 4.5%, both larger than the nitrogen and carbon percentagesof 0.2% and 3.3%, respectively, in the central parts of the uncoatedmagnetosomes, suggesting the presence of the coating. In a second seriesof measurements, when a suspension comprising the central parts of themagnetosomes coated with PEI is mixed with GL-261 cells and subjected totreatment condition 3, the temperature of the mixture increases from 37°C. before application of the field to 43° C. after 30 minutes ofapplication of the field. The initial slope of the temperature variationis estimated as 0.014° C./sec. When this same mixture is subjected totreatment condition 2, the percentage of living cells is 15% whereas itis 40% without application of the field (treatment condition 1). Thisindicates the cytotoxicity induced by the central parts of magnetosomescoated with PEI on GL261 cells in the presence of treatment condition 2.

Example 18 Central Parts of Magnetosomes Derived from AMB-1, Coated withPEI Preparation

A suspension comprising 10 mg in iron of the central parts of themagnetosomes, mixed with 10 mL of sterile non-pyrogenic water, issonicated for 5 minutes with the sonicating finger at 30 W with pulsesof 30 seconds, separated by intervals of 10 seconds. This suspension isplaced against a magnet, the supernatant is removed and then replacedwith 10 mL of a PEI solution at 20 mg/mL, mixed with sterilenon-pyrogenic water at pH 9.5. During this preparation, the mass of PEIis twice as high as the mass of iron. The obtained mixture is sonicatedfor 30 minutes with the sonicating finger at a power of 20 W with pulsesof 10 seconds, separated by intervals between pulses of 20 seconds. Thesuspension is cooled every two minutes in an ice bath. It is washed onceby placing a Neodinium magnet against the wall of the glass tube, byremoving the supernatant and then by replacing it with pycleroticHyClone water and by sonicating for 1 minute at 30 W. It is washed tenmore times in the same manner.

Characterization: The LAL test reveals an endotoxin concentration ofless than 50 EU/mg/mL in these suspensions. The TEM measurements enableto estimate the thickness of the coating surrounding the central partsof these magnetosomes as between 4 and 18 nm. The absorption of thesuspension of the central parts of magnetosomes coated with PEIcomprising 1 mg in iron, measured at 480 nm, decreased by 64% during the20 minutes of measurement. The light scattering measurements of thissuspension indicate the presence of objects of hydrodynamic size 125 nmfor 6% of them, 5445 nm for 1% of them and 1067 nm for 93% of them. Theisoelectric point is estimated at 11.3 and the zeta potential decreasesfrom 50 mV at pH 2 to −10 mV at pH 12. CHNS measurements reveal a carbonpercentage of 6.6%. When a suspension comprising the central parts ofmagnetosomes coated with PEI is mixed with GL-261 cells and subjected totreatment condition 3, the temperature of the mixture increases by 12°C. after 30 minutes of application of the field. The initial slope ofthe temperature variation is estimated as 0.04° C./sec. When this samemixture is subjected to treatment condition 2, the percentage of livingcells is 12% whereas it is 26% without application of the field(treatment condition 1). This indicates the cytotoxicity induced by thecentral parts of magnetosomes coated with PEI on GL261 cells in thepresence of treatment condition 2.

Example 19 Central Parts of Magnetosomes Derived from MSR-1, Coated withAl(OH)₃ Preparation

The suspension comprising 7 mg per milliliter of the central part of themagnetosomes is first sonicated for 5 minutes at a power of 5 W withpulses of 0.1 seconds and intervals between pulses of 0.1 seconds. Asuspension comprising 2 mg of the central part of the magnetosomes isintroduced in a 8 mL glass tube, the suspension is placed against aneodymium magnet, the supernatant is removed and replaced by 600 μl ofaluminum hydroxide at 10 mg/mL. The mixture is sonicated for 90 minutescontinuously at 20 W. A first wash is carried out by placing thesuspension against a magnet, by removing the supernatant and byreplacing it with HyClone water. Three additional washes were carriedout in the same manner.

Characterization: The absorption of the suspension comprising thecentral parts of the magnetosomes coated with Al(OH)₃, measured at 480nm, does not decrease during the 20 minutes of measurement, which showsthe stability of this suspension. TEM measurements reveal the presenceof a gel surrounding the central parts of these magnetosomes. The lightscattering measurements of this suspension indicate the presence ofobjects of hydrodynamic size 204 nm for 5% of them and 1810 nm for 95%of them. The isoelectric point is estimated to be pH 2.5 and the zetapotential decreases from 5 mV at pH 2 to −30 mV at pH 12. CHNSmeasurements reveal a carbon percentage of 3.3%. When a suspensioncomprising the central parts of the magnetosomes coated with Al(OH)₃ ismixed with GL-261 cells and subjected to treatment condition 3, thetemperature of the mixture increases by 21.3° C. after 30 minutes offield application. The initial slope of the temperature variation isestimated as 0.034° C./sec. When this same mixture is subjected totreatment condition 2, the percentage of living cells is 26% whereas itis 91% without application of the field (treatment condition 1). Thisindicates the cytotoxicity induced by the central parts of the centralparts of the magnetosomes coated with Al(OH)₃ on the GL261 cells in thepresence of treatment condition 2.

Example 20 Central Parts of Magnetosomes Derived from MSR-1, Coated withSilica (APTS)

Preparation: The suspension comprising 7 mg per milliliter of thecentral part of the magnetosomes is first sonicated for 5 minutes at apower of 5 W with pulses of 0.1 seconds and intervals between pulses of0.1 seconds. A suspension comprising 10 mg of the central part of themagnetosomes is introduced in a 8 mL glass tube, the suspension isplaced against a neodymium magnet, the supernatant is removed andreplaced with 2 mL of a mixture of hexane and absolute ethanol (1:1;v/v). The suspension is sonicated for a few minutes before addition of200 μL of APTS ((3-Aminopropyl) triethoxysilane, APTS; Sigma reference:440140), i.e. 211 mg and 500 μL of 5M NaOH. The mixture is sonicated for10 minutes at 85° C. at a power of 5 W with pulses of 0.1 seconds andintervals between pulses of 0.1 seconds. A first wash is carried out byplacing the suspension against a magnet, by removing the supernatant andby replacing it with a volume of 1.5 mL of a mixture of hexane andabsolute ethanol (1/1; v/v). Then the suspension is sonicated for 30seconds at a power of 5 W with pulses of 0.1 seconds and intervalsbetween pulses of 0.1 seconds. Three additional washes were carried outin the same manner. Then a volume of 1 milliliter of 5M sodium hydroxideis added to the suspension of nanoparticles and then the mixture issonicated for 15 minutes at 80° C. at a power of 5W with pulses of 0.1second and intervals between pulses of 0.1 seconds. A first wash iscarried out by placing the suspension against a magnet, by removing thesupernatant and by replacing it with HyClone water. Two additionalwashes were carried out in the same manner.

Characterization: The absorption of the suspension of the central partsof magnetosomes coated with silica comprising 1 mg of iron, measured at480 nm, decreases by 90% during the 20 minutes of measurement. TEMmeasurements reveal the presence of a gel surrounding the central partsof these magnetosomes. The light scattering measurements of thissuspension indicate the presence of objects of hydrodynamic size 235 nmfor 10% of them, 277 nm for 7% of them and 1986 nm for 93% of them. Theisoelectric point is estimated to be pH 6.7 and the zeta potentialdecreases from 39 mV at pH 2 to −31 mV at pH 12. CHNS measurementsreveal a carbon percentage of 7.4%. When a suspension comprising thecentral parts of the magnetosomes coated with silica is mixed withGL-261 cells and subjected to treatment condition 3, the temperature ofthe mixture increases by 26.9° C. after 30 minutes of application of thefield. The initial slope of the temperature variation is estimated to be0.1° C./sec. When this same mixture is subjected to treatment condition2, the percentage of living cells is 2.5% whereas it is 40% withoutapplication of the field (treatment condition 1). This indicates thecytotoxicity induced by the central parts of the magnetosomes coatedwith silica on GL261 cells in the presence of treatment condition 2.

Example 21 Different Coating Protocols

From the protocols described in Examples 6 to 20, it may be possible tocoat the central part of the magnetosomes with coating agentspoly-L-lysine, chitosan, carboxy-methyl-dextran, citric acid, oleicacid, silica, folic acid, DOPC, alendronate, neridronate, PEI, Al(OH)₃using for the mixture of the central parts and the coating agent a ratiobetween coating mass and central part mass between 10⁻⁹ and 10⁹, 10⁻⁶and 10⁶, or between 10⁻² and 10², a sonication time between 10⁻⁹ and 10⁹seconds, between 10⁻⁶ and 10⁶ seconds, or between 10⁻² and 10² secondsor a sonication power between 10⁻⁹ and 10⁹ W, between 10⁻⁶ and 10⁶ W, orbetween 10⁻² and 10² W.

Example 22 Binding Properties Between the Central Parts

TEM measurements carried out on central parts coated with differentcoatings (PEI, DOPC, Neridronate, Chitosan, citric acid, dextran, AlOH₃,silica, folic acid) reveal that the distance separating the outersurface of two central parts separated by bonding material is between 0and more than 400 nm, that the number of central parts linked togetherby bonding material is between 2 and more than 10,000, that the centralparts linked together by bonding material form different shapes such aschains, circles, rhombuses, quadrilaterals (table 3), that the chainsare characterized by the presence of different central parts whosefacets are parallel, suggesting an alignment of the crystallographicaxes of the different central parts in the direction of elongation ofthe chains.

TABLE 1 Pyrogenicity Stability Heating properties and anti-tumor (LALtest) (Δabs at activity (in vitro) Endotoxin 480 nm) % living % livingProperties of the samples concentration Δabs ΔT δT/δt cells cells SampleCoating Species (UE/mg/mL) (%) (° C.) (°C. · s⁻¹) (−B) (+B) BNF-Starchhydroxyethyl starch chemical <50  0 6.2 0.009  7 ± 5 78 ± 5 BNF-Starchhydroxyethylstarch chemical 0 7.6 0.01 86 31 Whole bacteria AMB-1 Wholebacteria MSR-1 Pyrogenic bacterial membrane AMB-1 18000-150000 30 20.50.043 Extracted chains Pyrogenic extracted bacterial membrane AMB-1 9.80.012 55 10 chains Pyrogenic extracted bacterial membrane MSR-12000-11300 9.4 0.019 39 ± 5  5 ± 5 chains Pyrogenic extracted Bacterialmembrane MSR-1 2000-17000 86 0.012 39 12 Chains Central parts of NoneAMB-1 80-90 magnetosomes Central parts of None AMB-1 20 to 100 66-9323.8 0.024 30 48 magnetosomes Central parts of None MSR-1 10 to 10060-80 magnetosomes Central parts of None MSR-1 9.8 0.012 77 0 to 9magnetosomes Central parts of Poly-L-lysine MSR-1 78 50 11 0.024 53 ± 523 ± 5 magnetosomes Central parts of Poly-L-lysine MSR-1 30 magnetosomesCentral parts of Chitosan MSR-1 3 8.5 0.009 20 ± 5 0 à 5 magnetosomesCentral parts of Chitosan MSR-1 25 0.014 magnetosomes Central parts ofCarboxy- MSR-1 0 28.8 0.023 63 ± 5 11 ± 5 magnetosomes méthyldextranCentral parts of Carboxy- MSR-1 10 magnetosomes méthyldextran Centralparts of Citric acid MSR-1 19 15 25.2 0.038 57 ± 5 26 ± 5 magnetosomesCentral parts of Citric acid MSR-1 57  7 magnetosomes Central parts ofOléic add MSR-1 0 28.4 0.051 magnetosomes Central parts of Oleic acidMSR-1 0 5 0.012 53 14 magnetosomes Central parts of Silica MSR-1magnetosomes Central parts of Silica MSR-1 90 26.9 0.100 40   2.5magnetosomes Central parts of Folic add MSR-1 0 20 0.042 93 ± 5  9 ± 5magnetosomes Central parts of DOPC MSR-1 0 33.5 0.049 13 ± 5 0 à 5magnetosomes Central parts of DOPC MSR-1 2-70 magnetosomes Central partsof DOPC AMB-1 magnetosomes Central parts of Alendronate AMB-1 53-144magnetosomes Central parts of Alendronate AMB-1 1 18.3 0.028 17  2magnetosomes Central parts of Neridronate MSR-1 0 6.7 0.017 44 ± 5 28 ±5 magnetosomes Central parts of PEI MSR-1 0 6 0.014 40 ± 5 19 ± 5magnetosomes Central parts of PEI AMB-1 <50  magnetosomes Central partsof PEI AMB-1 64 12 0.040 26 12 magnetosomes Central parts of Al(OH)₃MSR-1 0 magnetosomes Central parts of Al(OH)₃ MSR-1 21.3 0.034 91 26magnetosomes

TABLE 2 Hydro- Iso- dynamic Coating elec- size of thick- tric popu- Zetapotential (mV) CHNS analysis Sample properties ness point lation pH pHpH pH pH pH % % % % Sample type Coating Species (nm) (pH) (nm) 2 4 6 810 12 N C H S BNF-Starch starch BNF-Starch Hydroxyéthyl- chemical 1 to 49.5 117 7 6 6 5 −3 −20 8.7 starch Whole bacteria AMB-1 Whole bacteriaAMB-1 32.0  Whole bacteria MSR-1 11 49   0.4 Pyrogenic Bacetrial AMB-1 1to 5 4.2 20 2.5 −18 −26 −34 −38 extracted membrane chains PyrogenicMembrane AMB-1 986 13.9  extracted bactérienne (81%) chains 4363  (14%)176  (5%) Pyrogenic Bacterial MSR-1 1 to 5 0.7 4.1 0.7 0.4 extractedmembrane chains Pyrogenic Bacterial MSR-1 6.4 2822  15 14 3 −15 −26 −3112.2  extracted membrane (82%) chains 535 (18%) Central parts of NoneAMB-1 0 4.9 38 40 −55 −56 −58 −60 magnetosomes Central parts of NoneAMB-1 752 4.9 magnetosomes (97%) 5253   (3%) Central parts of None MSR-10 3.5 3076 18 −8 −1.5 −27 −35 −45 0.2 33   0.5 0.002 magnetosomes (79%)677 (21%) Central parts of Poly-L-lysine MSR-1 4 to 16 8.7 2489  43 3524.5 5 −14 −34 0.4 3.6 0.6 0.03 magnetosomes (92%) 137  (8%) Centralparts of Chitosan MSR-1 6 magnetosomes Central parts of Chitosan MSR-111 1908  46 31 30 29 21 −55 3.2 magnetosomes (93%) 273  (7%) Centralparts of Carboxy- MSR-1 2 to 20 3.4 5124  20 −8 −25 −30 −31 −31 themagnetosomes méthyldextran  (6%) 1359  (79%) 331 (15%) Central parts ofCarboxy- MSR-1 3.7 magnetosomes méthyldextran Central parts of Citricacid MSR-1 1 à 15 3.7 788 25 −12 −18 −27 −31 −38 0.8 3.7 0.3 0magnetosomes Central parts of Oleic acid MSR-1 0.5 to 5 3.5 123 30 −10−40 −50 −55 −60 magnetosomes ND ND −10 −30 −35 −35 Central parts ofOleic acid MSR-1 3.4 magnetosomes Central parts of Silica MSR-1 Gelmagnetosomes Central parts of Silica MSR-1 Gel 6.7 1986  39 20 5 −10 −25−31 7.4 magnetosomes (93%) 277  (7%) 235 (10%) Parties centrales Folicadd MSR-l lto4 7.9 2876  45 33 24.4 −0.2 −32 −43 des magnétosomes (90%)235 (10%) Central parts of Folic acid MSR-1 3.9 magnetosomes Centralparts of DOPC MSR-1 0.6 to 3 3 1871  10 −6.5 −16 −20 −25 −35magnetosomes (87%) 278 (13%) Central parts of DOPC MSR-1 7.5magnetosomes Central parts of DOPC AMB-1 50 à 150 magnetosomes Centralparts of Alendronate AMB-1 magnetosomes Central parts of AlendronateAMB-1 Matrix 3.5 2735  25 −7 −30 −40 −45 −47 9   magnetosomes (86%) 527(14%) Central parts of Neridronate MSR-1 19 to 200 3.5 5560  40 −7.9 −26−30 −31 −42 magnetosomes  (1%) 710 (59%) 207 (40%) Central parts ofNeridronate MSR-1 18.1  magnetosomes Central parts of PEI MSR-1 8 to 1011 175 42 39 37 29 8 −16 1.1 4.5 0.7 0 magnetosomes Central parts of PEIAMB-1 4 to 18 magnetosomes Central parts of PEI AMB-1 4 to 18 11.3 1067 50 44 35 26 12 −10 6.6 magnetosomes (93%) 5445   (1%) 125  (6%) Centralparts of Al(OH)₃ MSR-1 Gel magnetosomes Central parts of Al(OH)₃ MSR-1Gel 2.5 1810  5 −7 −16 −23 −26 −30 3.3 magnetosomes (95%) 204  (5%)

TABLE 3 Distance between the external surfaces of Number of central twocentral parts parts linked separated by binding together by bindingBacteria material material Coating species Min Max Min Max Geometricshape PEI MSR1 0 16 2 26 Individual chains, chains sticked together,chains, spherical aggregates, circles. PEI AMB1 0 191 2 5 Chains,circles DOPC MSR1 0 27 2 16 Chains, circles, diamond Neridronate MSR1 031 2 18 Chains, chains sticked together Chitosan MSR1 0 11 2 42 Chains,chains sticked together Citric acid MSR1 0 16 2 26 Chaines, circles,triangle, quadrilateral Dextran MSR1 0 113 2 8 Circles, chains AlOH₃MSR1 0 >200 nm NA >10000 Chain, circle in a gel Silica MSR1 0  >50 nmNA >10000 Chain, circle in a gel Folic acid MSR1 0 >400 nm NA >10000Chain, circle in a gel

The invention claimed is:
 1. A preparation comprising at least onesynthetic nanoparticle, the at least one synthetic nanoparticlecomprising: a crystallized mineral central part comprising predominantlyan iron oxide, the central part having been produced by a livingorganism, and a coating comprising materials not produced by said livingorganism, the coating covering the central part completely or partly,wherein the coating comprises less than 12% in mass of non-denaturedproteins from said living organism relative to the mass of the at leastone synthetic nanoparticle, and wherein the coating does not comprise alipid bilayer that completely covers the central part and thatoriginates from said living organism.
 2. The preparation according toclaim 1, wherein the living organism is a bacterium.
 3. The preparationaccording to claim 2, wherein the bacterium is a magnetotacticbacterium.
 4. The preparation according to claim 1, wherein the centralpart comprises maghemite and/or magnetite.
 5. The preparation accordingto claim 1, wherein the at least one synthetic nanoparticle isnon-pyrogenic.
 6. The preparation according to claim 1, wherein the atleast one synthetic nanoparticle further comprises proteins, and theproteins that are not in the coating are non-denatured proteins.
 7. Thepreparation according to claim 1, wherein the coating comprises 0% inmass of non-denatured proteins from said living organism relative to themass of the at least one synthetic nanoparticle.
 8. The preparationaccording to claim 1, wherein the at least one synthetic nanoparticlecomprises no endotoxins.
 9. The preparation according to claim 1,wherein the at least one synthetic nanoparticle comprises an endotoxinquantity, which abides by at least one quality or regulatory standard inforce applicable to medical devices, drugs or cosmetic products, inparticular conforming to an ISO quality standard or a currentpharmacopoeia.
 10. The preparation according to claim 1, wherein thecoating of the at least one synthetic nanoparticle enables anarrangement in a chain of at least two synthetic nanoparticles, said atleast two synthetic nanoparticles having crystallographic axesorientated in the direction of elongation of the chain.
 11. Thepreparation according to claim 1, wherein the coating comprises at leastone compound able to establish weak interactions or covalent bonds withthe central part of the at least one synthetic nanoparticle.
 12. Thepreparation according to claim 1, wherein the coating comprises at leastone compound able to establish interactions or bonds with Fe²⁺ or Fe³⁺ions, hydroxyls OH⁻, oxides 0²⁻, or crystalline defects of the centralpart.
 13. The preparation according to claim 1, wherein the central parthas a size comprised between 1 nm and 2 μm.
 14. The preparationaccording to claim 1, wherein the coating has a thickness larger than0.1 nm.
 15. The preparation according to claim 1, wherein the coatingcomprises at least one compound selected from the group consisting of achelator, an amphipathic molecule, a polarized or charged polymer, ametal or silicon oxide, a hydroxide of a metal or silicon, an acid, anacidic, basic, oxidized, reduced, neutral, positively charged,negatively charged derivative of these compounds, and a combination ofseveral of these compounds or derivatives.
 16. The preparation accordingto claim 1, wherein the coating comprises at least one compound selectedfrom the group consisting of a polysaccharide, a fatty acid, aphospholipid, a polymer of amino acids, polymeric or non-polymericsilica, and an aliphatic amino polymer, of an acidic, basic, oxidized,reduced, neutral, positively charged, negatively charged derivatives ofthese compounds and a combination of several of these compounds orderivatives.
 17. The preparation according to claim 1, wherein thecoating comprises at least one function selected from the groupconsisting of carboxylic acids, phosphoric acids, sulfonic acids,esters, amides, ketones, alcohols, phenols, thiols, amines, ether,sulfides, acid anhydrides, acyl halides, amidines, nitriles,hydroperoxides, imines, aldehydes, peroxides, of an acidic, basic,oxidized, reduced, neutral, positively charged, negatively chargedderivative of these compounds, and a combination of several of thesecompounds or derivatives.
 18. The preparation according to claim 1,wherein the coating comprises carbonaceous compounds.
 19. Thepreparation according to claim 18, wherein the at least one syntheticnanoparticle is prepared by a process comprising the steps of: obtainingor starting from at least one natural nanoparticle produced by a livingorganism, the at least one natural nanoparticle comprising acrystallized mineral central part that comprises predominantly an ironoxide and an original surrounding coating that covers the central part;removing from the central part at least a portion of the originalsurrounding coating; and applying a new coating not produced by theliving organism to the central part from which the at least a portion ofthe original surrounding coating has been removed, the new coatingcovering the central part completely or partly, to form the at least onesynthetic nanoparticle, which comprises a coating that comprises lessthan 12% in mass of non-denatured proteins originating from the livingorganism that produced the central part relative to the mass of the atleast one synthetic nanoparticle, and wherein the coating does notcomprise a lipid bilayer that completely covers the central part andthat originates from said living organism.
 20. A method for treating atumor in an individual or animal, wherein a therapeutically activequantity of the preparation according to claim 1 is administered to theindividual or animal.
 21. A pharmaceutical composition or drugcomprising, as active principle, a preparation as defined in claim 1,and optionally at least one pharmaceutically acceptable carrier.
 22. Amedical device comprising a preparation as defined in claim
 1. 23. Adiagnostic composition comprising a preparation as defined in claim 1.24. A cosmetic composition comprising, as cosmetic active principle, apreparation as defined in claim
 1. 25. A method for the manufacture of apreparation as defined in claim 1, comprising the following steps: (i)from a preparation of nanoparticles synthesized by a living organismcomprising a crystallized central part composed predominantly of an ironoxide and a biological surrounding coating, isolating the central part;(ii) treating the resulting isolated central part to cover the centralpart with a coating; (iii) optionally sterilize the preparation, afterstep (i) or after step (ii).